U.S. patent application number 10/565240 was filed with the patent office on 2007-12-20 for multiwell plate.
Invention is credited to Assaf Deutsch, Mordechai Deutsch, Max Herzberg, Reuven Tirosh.
Application Number | 20070292837 10/565240 |
Document ID | / |
Family ID | 38862014 |
Filed Date | 2007-12-20 |
United States Patent
Application |
20070292837 |
Kind Code |
A1 |
Deutsch; Mordechai ; et
al. |
December 20, 2007 |
Multiwell Plate
Abstract
A mulitwell plate having a plurality of picowells on the bottom
of the wells of the plate as well as methods of producing the
mulitwell plate are provided. Provided is also a method of handling
living cells by providing an ordered array of living cells
immobilized in a non-fluid matrix, contacting the living cells with
a stimulus; and detecting a response to the stimulus. The present
invention is also of a method of producing an ordered array of
living cells.
Inventors: |
Deutsch; Mordechai; (Doar-Na
Lev HaSharon, IL) ; Herzberg; Max; (Emeq Soreg,
IL) ; Tirosh; Reuven; (Kfar-Saba, IL) ;
Deutsch; Assaf; (Moshav-Tzfaria, IL) |
Correspondence
Address: |
Martin D. Moynihan;PRTSI, Inc.
P.O. Box 16446
Arlington
VA
22215
US
|
Family ID: |
38862014 |
Appl. No.: |
10/565240 |
Filed: |
July 20, 2004 |
PCT Filed: |
July 20, 2004 |
PCT NO: |
PCT/IL04/00661 |
371 Date: |
May 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60488408 |
Jul 21, 2003 |
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60488409 |
Jul 21, 2003 |
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60517073 |
Nov 5, 2003 |
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60517084 |
Nov 5, 2003 |
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60544356 |
Feb 17, 2004 |
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60544357 |
Feb 17, 2004 |
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Current U.S.
Class: |
435/4 ; 29/592;
422/400; 435/174; 435/307.1 |
Current CPC
Class: |
B01L 2300/0829 20130101;
B01L 3/5085 20130101; C12M 23/12 20130101; B01L 2400/0457 20130101;
B01L 2300/04 20130101; B29C 65/002 20130101; Y10T 29/49 20150115;
B29L 2009/00 20130101; B01L 2400/0487 20130101; C12N 11/04
20130101; G01N 33/5008 20130101; B01L 2200/0668 20130101; B01L
2400/0409 20130101; B29L 2031/712 20130101; B01L 2300/0896
20130101; B01L 2300/069 20130101; B01L 2400/0454 20130101; B29C
39/026 20130101; B29K 2101/12 20130101; B01L 2200/12 20130101; B01L
2300/0819 20130101; B29C 65/48 20130101; C12M 23/20 20130101; B01L
2300/0893 20130101 |
Class at
Publication: |
435/004 ;
029/592; 422/102; 435/174; 435/307.1 |
International
Class: |
C12Q 1/00 20060101
C12Q001/00; B01L 3/00 20060101 B01L003/00; B23P 17/04 20060101
B23P017/04; C12M 3/00 20060101 C12M003/00; C12N 11/00 20060101
C12N011/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 27, 2004 |
IL |
PCT/IL04/00571 |
Claims
1. A multiwell plate comprising a plurality of wells wherein at the
bottom surface of at least one well of said plurality of wells is a
plurality of picowells.
2. The plate of claim 1, having a footprint of a standard multiwell
plate.
3. The plate of claim 1, wherein said plurality of wells comprises
6n wells arranged in a 2n.times.3n array, where n is an integer
greater than 0.
4. (canceled)
5. The plate of claim 3, wherein said plurality of wells is
selected from the group consisting of 6, 24, 96, 384 and 1536
wells.
6-10. (canceled)
11. The plate of claim 1, wherein picowells of said plurality of
picowells are juxtaposed.
12. The plate of claim 11, wherein the interwell area between two
said picowells is less then about 0.35 the sum of the areas of said
two picowells.
13-16. (canceled)
17. The plate of claim 11, wherein a rim of a said picowell is
substantially knife-edged.
18. The plate of claim 1, wherein said plurality of picowells
comprises picowells having dimensions of less than about 200
microns.
19-24. (canceled)
25. The plate of claim 1, wherein picowells of said plurality of
picowells comprise enclosures of dimensions such that substantially
an entire cell of a certain size is containable within a said
enclosure, each said enclosure having an opening, said opening
defined by a first cross section of a size allowing passage of a
cell of a certain size.
26-37. (canceled)
38. The plate of claim 1, said plurality of picowells comprising
picowells, wherein all picowells of the plate are substantially
identical in size.
39. The plate of claim 1, wherein a first said well includes a
first said plurality of picowells and a second said well includes a
second said plurality of picowells, wherein said first plurality of
picowells and said second plurality of picowells are substantially
different.
40-42. (canceled)
43. The plate of claim 1, wherein the walls of wells of said
plurality of wells are integrally formed with said bottom
surface.
44. The plate of claim 1, further comprising at least one distinct
well-wall component attached to said bottom surface.
45. (canceled)
46. The plate of claim 1, wherein said plurality of picowells are
integrally formed with said bottom surface.
47. The plate of claim 1, further comprising at least one distinct
picowell-bearing component bearing said plurality of picowells
attached to said bottom surface of said one well.
48-50. (canceled)
51. The plate of claim 1, further comprising at least one distinct
picowell-bearing component bearing said plurality of picowells
resting within said one well.
52-54. (canceled)
55. The plate of claim 51, wherein said picowell-bearing component
comprises a gel.
56-57. (canceled)
58. The device of claim 55, wherein the water content of said gel
is greater than about 80% by weight.
59-63. (canceled)
64. The device of claim 55, wherein said gel comprises an active
entity.
65-72. (canceled)
73. The plate of claim 1, said plurality of picowells comprising
picowells, the bottom of said picowells substantially having an
index of refraction similar to that of water.
74. The plate of claim 73, wherein said index of refraction is less
than about 1.4.
75-80. (canceled)
81. The plate of claim 1, further comprising a gel cover covering
said plurality of picowells.
82. The plate of claim 1, wherein said plurality of picowells
covers substantially the entire said bottom surface of said
well.
83-87. (canceled)
88. A method of making a multiwell plate of claim 1, comprising:
(a) contacting a precursor material with a template including a
negative of features of the plate so as to create said features in
said precursor material, said features including said plurality of
picowells; (b) fixing said features in said precursor material so
as to fashion an incipient plate; and (c) processing said incipient
plate so as to fashion the plate.
89-92. (canceled)
93. The method of claim 88, further comprising: (d) prior to (a),
placing said precursor material in a well of a multiwell plate.
94. The method of claim 88, further comprising: (d) subsequent to
(b), attaching walls of said plurality of wells to said incipient
plate.
95-97. (canceled)
98. The method of claim 88, wherein said precursor material
includes a irreversibly deformable precursor material and said
fixing said features comprises separating said template from said
precursor material.
99. (canceled)
100. The method of claim 88, wherein said precursor material
comprises an reversibly deformable precursor material.
101-119. (canceled)
120. A method of making a multiwell plate of claim 1, comprising:
(a) placing a photoresist material on a precursor plate; and (b)
fixing a plurality of picowells in said photoresist material.
121-122. (canceled)
123. The method of claim 120, wherein said precursor plate
comprises a multiwell plate.
124-125. (canceled)
126. A method of making a multiwell plate of claim 1, comprising
placing a picowell-bearing component on a precursor plate.
127-134. (canceled)
135. A device comprising an array of living cells held in a
non-fluid matrix, said matrix configured to maintain cell
viability.
136. The device of claim 135, wherein said living cells are
physically held in pockets in said matrix.
137. (canceled)
138. The device of claim 135, wherein said array is substantially
planar having an upper surface and a lower surface.
139-144. (canceled)
145. The device of claim 135, said matrix comprising a material
having an index of refraction substantially similar to that of
water.
146. The device of claim 145, said matrix comprising a material
having an index of refraction less than about 1.4.
147-151. (canceled)
152. The device of claim 135, said matrix made of a material
comprising a gel.
153-156. (canceled)
157. A method for handling living cells, comprising: (a) providing
an ordered array of living cells immobilized in a non-fluid matrix,
said matrix configured to maintain cell viability; (b) contacting
said living cells with a stimulus; and (c) detecting a response of
said cells to said stimulus.
158-168. (canceled)
169. A method of producing an ordered array of living cells in a
non-fluid matrix, comprising: (a) providing a multiwell plate
provided with a plurality of wells, said multiwell plate including
a plurality of picowells at the bottom of at least one said well,
said plurality of picowells including picowells; (b) placing a
suspension of a plurality of living cells in a gellable fluid in
said at least one well; (c) causing said living cells to settle
into said picowells so as to be held in respective picowells; and
(d) gelling said gellable fluid so as to make a gel cover, trapping
said living cells between said picowells and said gel cover.
170. The method of claim 169, wherein said picowells are made of a
material comprising a gel.
171-175. (canceled)
176. The method of claim 169, wherein (e) prior to (d), ensuring
that substantially each picowell holds no more than one living
cell.
177-185. (canceled)
Description
FIELD AND BACKGROUND OF THE INVENTION
[0001] The present invention relates to the field of cellular
biology and more particularly, to an improved device and method for
the study of cells. Specifically, the present invention is of an
improved multiwell plate and methods for making the same that
allows the use of automatised sample handling methods for the study
of single living cells. Further, the present invention is of a
device substantially being an ordered array of living cells.
[0002] Combinatorial methods in chemistry, cellular biology and
biochemistry are essential for the near simultaneous preparation of
multitudes of active entities such as molecules. Once such a
multitude of molecules is prepared, it is necessary to study the
effect of each one of the active entities on a living organism.
[0003] The study of the effects of stimuli, such as exposure to
active entities, on living organisms is preferably initially
performed on living cells. Since, cell-functions include many
interrelated pathways, cycles and chemical reactions, the study of
an aggregate of cells, whether a homogenous or a heterogeneous
aggregate, does not provide sufficiently detailed or interpretable
results: rather a comprehensive study of the biological activity of
an active entity may be advantageously performed by examining the
effect of the active entity on a single isolated living cells.
Thus, the use of single-cell assays is one of the most important
tools for understanding biological systems and the influence
thereupon of various stimuli such as exposure to active
entities.
[0004] The combinatorial preparation of a multitudes of active
entities coupled with the necessity of studying the effect of each
one of the active entities on living organisms using a single-cell
assay, requires the development of high-throughput single live cell
assays.
[0005] In the art, various different methods for studying living
cells are known.
[0006] Multiwell plates having 6, 12, 48, 96, 384 or even 1536
wells on a standard ca. 8.5 cm by ca. 12.5 cm footprint are well
known in the art. Such multiwell plates are provided with an 2n by
3n array of rectangular packed wells, n being an integer. The
diameter of the wells of a plate depends on the number of wells and
is generally greater than about 250 microns (for a 1536 well
plate). The volume of the wells depends on the number of wells and
the depth thereof but generally is greater than 5.times.10.sup.-6
liter (for a 1536 well plate).
[0007] Multiwell plates are commercially available from many
different suppliers. Multiwell plates made from many different
materials are available, including but not limited to glass,
plastics, quartz and silicon. Multiwell plates having wells where
the inside surface is coated with various materials, such as active
entities, are known.
[0008] The standardization of the formats of multiwell plates is a
great advantage for researchers as the standardization allows the
production of standardized products including robotic handling
devices, automated sample handlers, sample dispensers, plate
readers, observation devices, plate washers, software and such
accessories as multifilters.
[0009] Although exceptionally useful for the study of large groups
of cells, multiwell plates are not suitable for the study of
individual cells or even small groups of cells due to the large,
relative to the cellular scale, size of the wells. Cells held in
such wells either float about a solution or adhere to a well
surface. When cells float about in a well, specific individual
cells are not easily found for observation. When cells adhere to a
well surface, the cells adhere to any location in the well,
including anywhere on the bottom of the well and on the walls of
the well. Such variability in location makes high-throughput
imaging (for example for morphological studies) challenging as
acquiring an individual cell and focusing thereon is extremely
difficult. Such variability in location also makes high-throughput
signal processing (for example, detection of light emitted by a
single cell through fluorescent processes) challenging as light
must be gathered from the entire area of the well, decreasing the
signal to noise ratio. Further, a cell held in a well of a
multiwell plate well can be physically or chemically manipulated
(for example, isolation or movement of a single selected cell or
single type of cell, changing media or introducing drugs) only with
difficulty. Further, the density of cells held singly in the wells
of a multiwell plate is very low (about 1536 cells in 65 cm.sup.2,
or 24 cells cm.sup.-2) Thus, multiwell plates are in general only
suitable for the study of homogenous or heterogenous aggregates of
cells as a group.
[0010] An additional disadvantage of multiwell plates is during the
study of cells undergoing apoptosis. One method of studying cells
is by exposing cells in a monolayer of cells adhered to the bottom
of the well of a multiwell plate to a stimulus. As is known
undergoes is apoptosis and it is highly desirable to observe a cell
throughout the apoptosis process. However, once a cell begins the
apoptosis process, the adhesion of the cell to the bottom of the
well is no longer sufficient: the cell detaches from the bottom and
is carried away by incidental fluid currents in the well. The cell
is no longer observable and its identity lost.
[0011] In the art, a number of method and devices have been
developed for the study of individual cells or a small number of
cells as a group. Many such methods are based on using
picowell-bearing devices. A picowell-bearing device is a device for
the study of cells that has at least one picowell-bearing component
for study of cells. A picowell-bearing component is a component
having at least one, but generally a plurality of picowells, each
picowell configured to hold at least one cell. The term "picowell"
is general and includes such features as dimples, depressions,
tubes and enclosures. Since cells range in size from about 1
microns to about 100 (or even more) microns diameter there is no
single picowell size that is appropriate for holding a single cell
of any type. That said, the dimensions of the typical individual
picowell in the picowell-bearing components known in the art have
dimensions of between about 1 microns up to about 200 microns,
depending on the exact implementation. For example, a device
designed for the study of single isolated 20 micron cells typically
has picowells of dimensions of about 20 microns. In other cases,
larger picowells are used to study the interactions of a few cells
held together in one picowell. For example, a 200 micron picowell
is recognized as being useful for the study of the interactions of
two or three cells, see PCT patent application IL01/00992 published
as WO 03/035824.
[0012] One feature that increases the utility of a picowell-bearing
device is that each individual picowell is individually
addressable. By individual addressability is meant that each
picowell can be registered, found or studied without continuous
observation. For example, while cells are held in picowells of a
picowell-bearing component, each cell is characterized and the
respective picowell where each cell is held is noted. When desired,
the observation component of the picowell-bearing device is
directed to the location of the picowell where a specific cell is
held. One method of implementing individual addressability is by
the use of fiducial points or other features (such as signs or
labels), generally on the picowell-bearing component. Another
method of implementing individual addressability is by arranging
the picowells in a picowell-array and finding a specific desired
picowell by counting. Another method of implementing individual
addressability is by providing a dedicated observation component
for each picowell.
[0013] In the art, the picowell-bearing component of
picowell-bearing devices is often a chip, a plate or other
substantially planar component. Herein such a component is termed a
"carrier". In the art, there also exist non-carrier
picowell-bearing components of picowell-bearing devices, for
example, bundles of fibers or bundles of tubes.
[0014] Mrksich and Whitesides, Ann. Rev. Biophys. Biomol. Struct.
1996, 25, 55-78; Craighead et al., J. Vac. Sci. Technol. 1982, 20,
316; Singhvi et al., Science 1994, 264, 696-698; Aplin and Hughes,
Analyt. Biochem. 1981, 113, 144-148 and U.S. Pat. No. 5,324,591 all
teach of devices including arrays of spots of cell-attracting or
cell-binding entities on a plate. In such devices, the spots serve
as picowells, binding to cells through a variety of chemical bonds.
In such devices, the plate is the picowell-bearing component of the
device. Due to the size of the spots, each such picowell generally
holds more than one cell. To reduce interaction between cells held
at different picowells, the spots must be spaced relatively far
apart, reducing loading as expressed in terms of picowells per unit
area. Even with generous spacing, in such picowell-bearing
components held cells are not entirely isolated from mutual
interaction, nor can cells be subject to individual manipulation.
The fact that the cells are not free-floating but are bound to the
plate through some interaction necessarily compromises the results
of experiments performed.
[0015] In U.S. Pat. No. 6,103,479, the picowell-bearing component
is a transparent carrier provided with a non-uniform array of
picowells, each well functionalized with chemical entities that
bind to cells specifically or non-specifically. Each picowell is of
approximately 200 to 1000 micron diameter and is configured to hold
a plurality of cells. The inter picowell areas are hydrophobic so
as not to attract cells. In addition to the carrier, a device of
U.S. Pat. No. 6,103,479 is provided with a glass, plastic or
silicon chamber-bearing plate in which individually addressable
microfluidic channels are etched that mates with the carrier. When
brought together, the carrier and chamber-bearing plate constitute
a cassette in which each cell is bound to the carrier and isolated
in a chamber provided with an individual fluid delivery system.
Reagents are provided through the fluid delivery system and
observed by the detection of fluorescence. In order to provide
space for the walls of the chambers, the inter picowell areas of
the carrier are relatively large, reducing loading as expressed in
terms of picowells per unit area. Subsequent to study, the cassette
is separated into the two parts and the micro-patterned array of
cells processed further. In some embodiments, the chamber-bearing
plate is made of polytetrafluoroethylene, polydimethylsiloxane or
an elastomer. As held cells do not make contact with the
chamber-bearing plate it is not clear what advantages are to be had
when providing a chamber-bearing plate of such esoteric
materials.
[0016] In U.S. Pat. No. 4,729,949, a device is taught for trapping
individual cells in a picowell-bearing carrier, the carrier being
substantially a plate having a plurality of picowells that are
individually-addressable tapered apertures of a size to hold
individual cells. Suction applied from the bottom surface of the
plate where the picowells are narrow creates a force that draws
cells suspended in a fluid above the carrier into the wide end of
the picowells on the surface of the carrier to be held therein.
Using the teachings of U.S. Pat. No. 4,729,949 a specific group of
cells (having dimensions similar to that of the wide end of the
picowells) can be selected from amongst a group of cells and held
in the carrier. Although the cells are subjected to common stimuli,
the fact that the picowells are individually addressable allows the
effect of a stimulus on an individual cell to be observed. A
carrier of U.S. Pat. No. 4,729,949, is generally made of metal such
as nickel and prepared using standard photoresist and
electroplating techniques. In a carrier of U.S. Pat. No. 4,729,949,
the inter picowell areas of the carrier are relatively large,
leading to a low loading as expressed in terms of picowells per
unit area. Further, the suction required to hold cells in picowells
of a carrier of U.S. Pat. No. 4,729,949 deforms held cells and
makes a significant portion of the cell membranes unavailable for
contact, both factors that potentially compromise experimental
results. Study of cells with non-fluorescence based methods
generally gives poor results due to reflections of light from the
carrier.
[0017] In PCT patent application US99/04473 published as WO
99/45357 is taught a picowell-bearing device produced by etching
the ends of a bundle of optical fibers (apparently of glass) while
leaving the cladding intact to form a picowell-bearing component
that is a bundle of tubes. The size of the hexagonal picowells is
demonstrated to be as small as 7 micron wide, 5 micron deep and
having a volume of 1.45.times.10.sup.-13 liter. The inter picowell
area is quite large due to the thickness of the cladding of the
optical fibers. Cells held in each picowell are independently
observable through a respective optical fiber. In some embodiments,
the inside surface of the picowells is coated with a film of
materials such as collagen, fibronectin, polylysine, polyethylene
glycol, polystyrene, fluorophores, chromophores, dyes or a metal.
Loading the picowell-bearing component of PCT patent application
US99/04473 includes dipping the optical fiber bundle in a cell
suspension so that cells adhere to the picowells. There are a
number of disadvantages to the teachings of PCT patent application
US99/04473. The fact that the cells are studied only subsequent to
adhesion to the picowells necessarily influences the results of
experiments performed. Since cell proliferation generally begins
soon after adhesion, it is never clear if a signal detected results
from a single cell or a plurality of cells. It is not clear where
exactly in a picowell a cell is held and therefore what percentage
of light emitted from a cell travels to a detector. The fact that
emitted light travels through an optical fiber leads to loss of
time-dependent and phase information.
[0018] In unpublished copending PCT patent application IL04/00192
of the Applicant filed 27 Jun. 2004 is taught a picowell-bearing
device produced by bundling together glass capillaries, each glass
capillary attached to an independent fluid flow generator such as a
pump. A cell held in a first picowell is transferred to a second
picowell by the simultaneous application of an outwards flow from
the first picowell and an inwards flow into the second
picowell.
[0019] A preferred device for the study of cells is described in
PCT patent application IL10/00992 published as WO 03/035824. The
device 10, depicted in FIG. 1, is provided with a transparent
carrier 12 as a picowell-bearing component. Carrier 12 is
substantially a sheet of transparent material (such as glass or
polystyrene) on the surface of which features such as inlet
connectors 14, fluid channels 16, picowells (in FIG. 1a well-array
18), a fluid reservoir 20 and an outlet connector 22. Carrier 12 is
immovably held in a holder 24 having a cutout window of a size and
shape to accept carrier 12. Other components of device 10 not
depicted include flow generators, observation components, external
tubing and the like. When a cover slip (not depicted) is placed or
integrally formed with carrier 12, fluid channels 16,
picowell-array 18 and reservoir 20 are sealed forming channels that
allow transport of fluids and reagents to cells held in
picowell-array 18. The picowells are configured to hold a
predetermined number of cells (one or more) of a certain size and
are preferably individually addressable both for examination and
manipulation.
[0020] FIG. 2 is a reproduction of a photograph of a different
carrier 26 held in a holder 24. A first syringe 28 as an inlet flow
generator is in communication with an inlet connector 14 by a
capillary tube 30. Inlet connector 14 is in communication with
picowell-array 18 through a fluid passage 16. Picowell-array 18 is
in communication with outlet connector 22 through a fluid passage
16. A second syringe 32 as an outlet flow generator is in
communication with outlet connector 22 through capillary tube
34.
[0021] PCT patent application IL01/00992 also teaches methods of
physically manipulating cells held in a picowell-bearing device
using, for example, individually addressable microelectrodes (found
in the picowells or in the cover slip) or optical tweezers. Typical
physical manipulations include moving cells into or out of
picowells. One useful method that is implemented using a device of
PCT patent application IL01/00992 is that cells, each held alone in
a respective picowell, are examined (either in the presence or
absence of reagents) and based on the results of the examination,
cells with a certain characteristic are selected to remain in a
respective picowell while cells without the certain characteristic
are removed from a respective picowell and ejected by the
application of a flow in parallel to the surface of the carrier,
generated by a flow generator.
[0022] An additional feature of the teachings of PCT patent
application IL01/00992 is that, in some embodiments, the picowells
are juxtaposed, that is, the area occupied by a picowell-array is
substantially entirely made up of picowells with little or no inter
picowell area, see FIG. 3. FIG. 3 is a reproduction of a photograph
of part of a picowell-array 18 from the top of a carrier 12 of PCT
patent application IL01/00992. In FIG. 3 is seen a plurality of
hexagonal picowells 36, some populated with living cells 38. It is
seen that the inter picowell areas 40 make up only a minor
percentage of the total area of picowell-array 18. This feature
allows near tissue-density packing of cells, especially in
single-cell picowell configurations. For example, a typical device
of PCT patent application IL01/00992 having a 2 mm by 2 mm
picowell-array of hexagonally-packed juxtaposed picowells of 10
micron diameter and no inter picowell area includes about 61600
picowells. This feature also allows simple picowell loading: a
fluid containing suspended cells is introduced in the volume above
the picowells. Since there is little inter picowell area, cells
settle in the picowells.
[0023] Despite the utility of the device taught in PCT patent
applications IL01/00992, the use of the device is too labor
intensive for certain high-throughput implementations. Amongst
other reasons the large amount of labor is required because there
exist no commercially available robotic systems optimized for use
with the devices.
[0024] It would be highly advantageous to have a device for the
study of cells not having at least some of the disadvantages of the
prior art.
SUMMARY OF THE INVENTION
[0025] The present invention successfully addresses at least some
of the shortcomings of the prior art by providing an improved
multiwell plate and a new device, a method for producing the
improved multiwell plate and the new device and new methods for
handling living cells.
[0026] According to the teachings of the present invention there is
provided a multiwell plate comprising a plurality of wells wherein
at the bottom surface of at least one well of the plurality of
wells is a plurality of picowells. Preferably, a plate of the
present invention has a footprint of a standard multiwell plate.
Preferably, the plurality of wells of a plate of the present
invention comprises 6n wells arranged in a 2n by 3n array, where n
is an integer greater than 0, the wells preferably being arranged
in rectangular packing. Preferred pluralities of wells are the
commonly known pluralities of well such as 6, 24, 96, 384 and 1536
wells. Most preferred are plates of 96 wells and 384 wells as these
formats are most popular and have many available accessories
including fluid-handling accessories such as fluid-handling
robots.
[0027] In an embodiment of the present invention, the plurality of
picowells comprises individually addressable picowells. In an
embodiment of the present invention, the bottoms of all picowells
in a given well of a plate of the present invention are
substantially coplanar. In an embodiment of the present invention,
the bottoms of all picowells of a plate of the present invention
are substantially coplanar.
[0028] In an embodiment of the present invention, the picowells of
a plurality of picowells in a given well are juxtaposed. By
juxtaposed is meant that in an area where picowells are found, most
of the area is picowell area and little of the area is inter
picowell area. According to a feature of the present invention, by
juxtaposed is meant that the inter picowell area between two
picowells is less than or equal to 0.35, 0.25, 0.15, 0.10 or even
0.06 of the sum of the areas of the two picowells. In certain
embodiments of the present invention it is preferred that the inter
picowell area be substantially zero, that is that the rims of
picowells are substantially knife-edged.
[0029] The dimensions of picowells of a multiwell plate of the
present invention, depending on the specific embodiment, are less
than about 200 microns, less than about 100 microns, less than
about 50 microns, less than about 25 microns or even less than
about 10 microns. In an embodiment of the present invention,
picowells are configured to hold no more than one living cell of a
certain size at any one time. In an embodiment of the present
invention, picowells are configured to hold no more than a
predetermined number of living cells of a certain size at any one
time.
[0030] In an embodiment of the present invention, the picowells are
enclosures of dimensions such that substantially at least one
entire cell of a certain size is containable within such an
enclosure, each enclosure having an opening at the surface of the
carrier, the opening defined by a first cross section of a size
allowing passage of a cell of the certain size. Depending on the
embodiment, the volume of such an enclosure is typically less than
about 1.times.10.sup.-11 liter, less than about 1.times.10.sup.-12
liter, less than about 1.times.10.sup.-13 liter, less than about
1.times.10.sup.-14 liter or even less than about 1.times.10.sup.-5
liter. Depending on the embodiment, the area of the first cross
section of such an enclosure is typically less than about 40000
micron.sup.2, less than about 10000 micron.sup.2, less than about
2500 micron.sup.2, less than about 625 micron.sup.2 or even less
than about 100 micron.sup.2. In an embodiment of the present
invention, picowells enclosures are configured to hold no more than
one living cell of a certain size at any one time. In an embodiment
of the present invention, picowells enclosures are configured to
hold no more than a predetermined number of living cells of a
certain size at any one time.
[0031] In an embodiment of the present invention, the plurality of
picowells comprises picowells, wherein all picowells of the plate
are substantially identical in size.
[0032] In another embodiment of the present invention, a first well
of a plate of the present invention includes a first plurality of
picowells and a second well of a plate includes a second plurality
of picowells, wherein the first plurality of picowells and the
second plurality of picowells are substantially different. For
example, in an embodiment of the present invention the size of the
picowells of the first plurality is substantially different from
the size of picowells of the second plurality of picowells.
[0033] A multiwell plate of the present invention is made of any
suitable material. Suitable materials include but are not limited
to ceramics, elastomers, epoxies, glasses, glass-ceramics, metals,
plastics, polycarbonates, polydimethylsiloxane, polyurethane,
polyethylenterephtalate glycol, polymers, polymethyl methacrylate,
polystyrene, polyvinyl chloride, rubber, silicon, silicon oxide and
silicon rubber.
[0034] In an embodiment of the present invention, the bottom
surface of the wells is made of any suitable material. Suitable
materials include but are not limited to ceramics, elastomers,
epoxies, glasses, glass-ceramics, metals, plastics, polycarbonates,
polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
polymethyl methacrylate, polystyrene, polyurethane, polyvinyl
chloride, rubber, silicon, silicon oxide and silicon rubber.
[0035] In embodiments of the present invention, an entire plate of
the present invention and all components thereof are made of one
material. In other embodiments, a plate of the present invention is
made up of a number of different materials, for example, as a
plurality of layers or as a coated structure.
[0036] In an embodiment of the present invention, the walls of
wells of the plurality of wells are integrally formed with the
bottom surface of the wells.
[0037] In other embodiments, a plate of the present invention
comprises at least one distinct well-wall component attached to the
bottom surface. Such a distinct well-wall component is made of any
suitable material. Suitable materials include but are not limited
to ceramics, elastomers, epoxies, glasses, glass-ceramics, metals,
plastics, polycarbonates, polydimethylsiloxane,
polyethylenterephtalate glycol, polymers, polyurethane, polymethyl
methacrylate, polystyrene, polyvinyl chloride, rubber, silicon,
silicon oxide and silicon rubber.
[0038] In an embodiment of the present invention, a plurality of
picowells are integrally formed with the bottom surface.
[0039] In an embodiment of the present invention, a plate of the
present invention comprises at least one distinct picowell-bearing
component bearing a plurality of picowells, the component attached
to the bottom surface of a respective well or simply resting within
a respective well.
[0040] A suitable distinct picowell-bearing component is a carrier
comprising a plurality of picowells disposed on a surface, such as
a carrier described in PCT patent application IL01/00992 or in
unpublished copending PCT patent application IL04/00571 of the
Applicant filed 27 Jun. 2004 (vide infra). Picowell-bearing
components are made of any suitable material, including reversibly
deformable materials and irreversibly deformable materials.
Suitable materials include but are not limited to gels, hydrogels,
waxes, hydrocarbon waxes, crystalline waxes, paraffins, ceramics,
elastomers, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate
glycol, polymers, polymethyl methacrylate, polystyrene,
polyurethane, polyvinyl chloride, rubber, silicon, silicon oxide
and silicon rubber.
[0041] In an embodiment of the multiwell plate of the present
invention, the picowell-bearing component comprises a gel,
preferably a transparent gel, preferably a hydrogel.
[0042] Gels suitable for use in making a picowell-bearing component
of a plate of the present invention include but are not limited to
agar gels, agarose gels, gelatins, low melting temperature agarose
gels, alginate gels, room-temperature Ca.sup.2+-induced alginate
gels and polysaccharide gels. Depending on the embodiment, a
suitable gel has a water content of greater than about 80% by
weight, greater than about 92% by weight, greater than about 95% by
weight, greater than about 97% by weight and even greater than
about 98% by weight. In an embodiment of the present invention, the
gel includes an active entity. Suitable active entities include,
but are not limited to antibodies, antigens, biological materials,
chemical materials, chromatogenic compounds, drugs, enzymes,
fluorescent probes, immunogenes, indicators, ligands, nucleic
acids, nutrients, peptides, physiological media, proteins,
receptors, selective toxins and toxins.
[0043] In an embodiment of the present invention, picowells have a
bottom surface made of a first material and borders, such as the
borders delineating the picowells, made of a second material, the
second material being substantially different from the first
material. In an embodiment of the present invention the first
material is substantially the material from which the bottom of the
well is made, for example when the bottom surface of the picowell
is substantially the bottom surface of the well. In an embodiment
of the present invention, the second material is a fixed
photoresist material.
[0044] In an embodiment of the plate of the present invention, the
plurality of picowells comprises picowells having an inside surface
configured to delay proliferation of cells held therein, for
example, by delaying adhesion of living cells thereto. In an
embodiment of the plate of the present invention, the inside of a
picowell comprises a material that delays adhesion of living cells
thereto, that is the picowell is substantially fashioned from the
adhesion-delaying material or the inside of the picowell is coated
with the adhesion-delaying material. A suitable material to coat
the inside of a picowell or from which to make a picowell comprises
polydimethylsiloxane, is substantially polydimethylsiloxane or is
substantially pure polydimethylsiloxane.
[0045] In an embodiment of the present invention bottom surfaces of
picowells making up a plurality of picowells of a plate comprise a
material having an index of refraction similar to that of water. In
a preferred embodiment of a plate of the present invention, the
index of refraction of the bottom surfaces is less than about 1.4,
less than about 1.38, less than about 1.36, less than about 1.35,
less than about 1.34 or substantially equal to that of water.
[0046] In an embodiment of the present invention, the plurality of
picowells comprises picowells having an inner surface coated with a
layer of a material. Suitable materials for coating an inner
surface of a picowell of a plate of the present invention include
but are not limited to gels, hydrogels, polydimethylsiloxane,
elastomers, polymerized para-xylylene molecules, polymerized
derivatives of para-xylylene molecules, rubber and silicon
rubber.
[0047] In an embodiment of the present invention, a plate of the
present invention further comprises a gel cover covering a
plurality of the picowells, the cover made of a gel. Suitable gels
are as described hereinabove.
[0048] In an embodiment of the present invention, substantially the
entire bottom surface of a well is covered by a respective
plurality of picowells.
[0049] In an embodiment of the present invention, a plate further
comprises at least one additional feature functionally associated
with the plurality of picowells, especially microfluidic features.
Suitable microfluidic features include but are not limited to
channels, coupling elements, drains, fluid channels, fluid
reservoirs, input ports, membranes, microreactors, microvalves,
output ports, passages, plumbing routes, protruberances, pumps,
transport channels and valves. Other suitable features include but
are not limited to light sources, magnetizable elements, optical
components, optical fibers, optical filters, protuberances,
fiducial points and walls.
[0050] In an embodiment of the present invention, a plate further
comprises a cover slip, the cover slip and a plurality of picowells
in a well configured so as to allow the cover slip to rest above
the plurality of picowells substantially in parallel to the bottom
surface of the well.
[0051] According to the teachings of the present invention, there
is provided a method of making a multiwell plate of the present
invention, comprising: (a) contacting a precursor material with a
template including a negative of features of the plate so as to
create the features in the precursor material, the features
including the plurality of picowells; (b) fixing the features in
the precursor material so as to fashion an incipient plate; and (c)
processing the incipient plate so as to fashion the multiwell plate
of the present invention.
[0052] Depending on the embodiment and the nature of the precursor
material, fixing includes such methods a heating the precursor
material, cooling the precursor material, polymerizing the
precursor material, cross-linking the precursor material, curing
the precursor material, irradiating the precursor material,
illuminating the precursor material, gelling the precursor
material, exposing the precursor material to a fixative and waiting
a period of time.
[0053] The template is generally made of a material that is rigid
compared to the precursor material. Suitable materials include but
are not limited to reversibly deformable materials, irreversibly
deformable materials, ceramics, epoxies, glasses, glass-ceramics,
metals, plastics, polycarbonates, polydimethylsiloxane,
polyethylenterephtalate glycol, polymers, polymethyl methacrylate,
paraffins, polystyrene, polyurethanes, polyvinyl chloride, silicon,
silicon oxide, silicon rubbers and wax.
[0054] Features created in the precursor material in addition to
the plurality of picowells include such features as channels,
coupling elements, drains, fluid channels, fluid reservoirs, input
ports, light sources, magnetizable elements, membranes,
microreactors, microvalves, passages, optical components, optical
fibers, optical filters, output ports, plumbing routes,
protruberances, pumps, transport channels, valves, walls and
fiducial points. In an embodiment of the present invention, the
features created in the precursor material in addition to the
plurality of picowells include the plurality of wells.
[0055] In an embodiment of the present invention, prior to
contacting the template with the precursor material, the precursor
material is placed in a well of a preexisting multiwell plate.
[0056] In an embodiment of the present invention, subsequent to the
fixing of the features, walls of the plurality of wells are
attached to the incipient plate. Attaching includes the use of
methods employing adhesives or surface treatments such as plasma
treatments.
[0057] In an embodiment of the present invention the precursor
material is an irreversibly deformable material (vide infra) such
as a wax, a paraffin, plastic or polymer, and fixing the features
simply includes separating the template from the precursor
material.
[0058] In an embodiment of the present invention the precursor
material is a reversibly deformable material (vide infra) such as a
gellable fluid, a polymerizable material, a powder, a fluid or a
thermoplastic material.
[0059] In an embodiment of the present invention, the reversibly
deformable precursor material is a thermoplastic material at
plastic temperature and fixing the features includes cooling the
thermoplastic material.
[0060] In an embodiment of the present invention, the reversibly
deformable precursor material is a polymerizable material and
fixing the features includes polymerizing the polymerizable
material. Suitable polymerizable materials include but are not
limited to monomer solutions, crosslinkable polymers, vulcanizable
polymers, polymerizable fluid and thermosetting resins.
[0061] In a preferred embodiment, the polymerizable material is a
polydimethylsiloxane precursor mixture and fixing the features
includes polymerizing the polydimethylsiloxane precursor mixture so
as to produce polydimethylsiloxane. In another preferred
embodiment, the polymerizable material includes urethane and fixing
the features includes polymerizing the urethane to produce
polyurethane.
[0062] In an embodiment of the present invention, the reversibly
deformable precursor material is a gellable fluid and fixing the
features includes gelling the gellable fluid. Depending on the
nature of the gellable fluid used, preferred methods of gelling the
gellable fluid include of heating the gellable fluid, cooling the
gellable fluid, irradiating the gellable fluid, illuminating the
gellable fluid, contacting the gellable fluid with a gelling
reagent and waiting a period of time for the gellable fluid to gel.
Suitable gellable fluids include but are not limited to agars,
agaroses, gelatins, low melting temperature agaroses, alginates,
proteins, protein polysaccharides, room-temperature
Ca.sup.2+-inducable alginates and polysaccharides. A preferred
gellable fluid is an alginate solution where gelling the gellable
fluid includes contacting the gellable fluid with a gelling
reagent, such as a gelling reagent including Ca.sup.2+ ions. An
additional preferred gellable fluid is a low melting temperature
agarose solution and gelling the gellable fluid includes cooling
the gellable fluid.
[0063] In an embodiment of the present invention, processing the
incipient plate comprises coating an inside surface of picowells of
the plurality of picowells with a layer of a coating material.
[0064] According to the teachings of the present invention there is
provided an additional method of making a multiwell plate of the
present invention, comprising (a) placing a photoresist material on
a precursor plate; and (b) fixing a plurality of picowells in the
photoresist material. Preferably, the fixing of the plurality of
picowells comprises irradiating the photoresist material through a
mask. A precursor plate is made of a suitable material. Suitable
materials include but are not limited to ceramics, epoxies,
glasses, glass-ceramics, metals, plastics, polycarbonates,
polydimethylsiloxane, polymers, polyethylenterephtalate glycol,
polymethyl methacrylate, polystyrene, polyurethanes, polyvinyl
chloride, silicon and silicon oxide.
[0065] In an embodiment of the method of the present invention the
precursor plate comprises a multiwell plate. The photoresist
material is placed in a well of the precursor plate and the
photoresist material irradiated inside the well.
[0066] In an embodiment of the present invention, subsequent to the
fixing of the features, walls of the plurality of wells are
attached to the precursor plate. Attaching includes the use of
methods employing adhesives or surface treatments such as plasma
treatments.
[0067] In an embodiment of the present invention, subsequent to
fixing the picowells in the photoresist material, the inside
surface of picowell of the plurality of picowells is coated with a
layer of a coating material.
[0068] According to the teachings of the present invention, there
is provided an additional method for making a multiwell plate of
the present invention by placing a picowell-bearing component on a
precursor plate. In a preferred embodiment, the picowell-bearing
component is attached to the precursor plate. Attaching includes
the use of methods employing adhesives or surface treatments such
as plasma treatments. A suitable picowell-bearing component
includes a carrier comprising a plurality of picowells disposed on
a surface, such as a carrier described in PCT patent application
IL01/00992 or in unpublished copending PCT patent application
IL04/00571 of the Applicant filed 27 Jun. 2004 (vide infra).
[0069] In an embodiment of the method of the present invention the
precursor plate comprises a multiwell plate and the
picowell-bearing component is placed in a respective well.
[0070] In an embodiment of the present invention, subsequent to the
placing of the picowells-bearing component on the precursor plate,
walls of the plurality of wells are attached to the precursor
plate. Attaching includes the use of methods employing adhesives or
surface treatments such as plasma treatments.
[0071] In an embodiment of the present invention, subsequent to
placing the picowells on the precursor plate, the inside surface of
picowells of the plurality of picowells are coated with a layer of
a coating material.
[0072] As noted above, whatever method is used for making a
multiwell plate of the present invention, it is often desired to
coat the plurality of picowells, especially the inside surface of
picowells with some material. Coating the inside surface of a
picowell allows modification of the properties of the picowell, for
example to reduce cytotoxicity, to change physical properties such
as solvent resistance or permeability or to delay proliferation of
cells held in a respective picowell. In an embodiment of the method
of the present invention, coating the inside surface of picowells
comprises (i) applying a precursor fluid to inside surfaces of the
picowells; and (ii) solidifying the precursor fluid so as to form
the layer. Suitable methods of solidifying include but are not
limited to heating the precursor fluid, cooling the precursor
fluid, polymerizing the precursor fluid, cross-linking the
precursor fluid, curing the precursor fluid, irradiating the
precursor fluid, illuminating the precursor fluid, gelling the
precursor fluid, exposing the precursor fluid to a fixative and
waiting a period of time.
[0073] In another embodiment of the method of the present
invention, coating the inside of the wells comprises (i) depositing
a vapor of the coating material onto the inside surface of the
picowells thereby forming the layer of coating material.
[0074] In another embodiment of the present invention, coating the
inside surface of the wells comprises (i) depositing a vapor of a
coating precursor material onto the inside surface of the
picowells; and (ii) solidifying the coating precursor material
thereby forming the layer of the coating material. Suitable methods
of solidifying the coating precursor material depend on the details
of the specific embodiment and include but are not limited to
heating the coating precursor material, cooling the coating
precursor material, polymerizing the coating precursor material,
cross-linking the coating precursor material, curing the coating
precursor material, irradiating the coating precursor material,
illuminating the coating precursor material, gelling the coating
precursor material, exposing the coating precursor material to a
fixative and waiting a period of time. In a preferred embodiment,
the vapor of coating precursor material is a vapor of para-xylylene
molecules or derivatives thereof and the layer comprises the
polymerized para-xylylene molecules (or derivatives thereof). By
para-xylylene derivatives is meant a a molecule that is
substantially a para-xylylene molecules having any additional
substituent on either or both aromatic rings.
[0075] According to the teachings of the present invention there is
also provided a device comprising an array of living cells held in
a non-fluid matrix, where the matrix is configured to maintain cell
viability. Preferably, the living cells are physically held in
pockets in the matrix and there is substantially no bond between
the living cells and the matrix. In a preferred embodiment, the
array is substantially planar having an upper surface and a lower
surface. In a preferred embodiment, one or both of the two surfaces
is transparent to at least one wavelength of light or range of
wavelengths of light in the ultraviolet, visible or infrared light
spectrum.
[0076] In a preferred embodiment of the present invention, the
matrix is configured to substantially delay the proliferation of
living cells held therein.
[0077] In an embodiment of the device of the present invention the
matrix comprises a material having an index of refraction similar
to that of water. In a preferred embodiment of the device present
invention, the index of refraction of the matrix is less than about
1.4, less than about 1.38, less than about 1.36, less than about
1.35, less than about 1.34 or substantially equal to that of
water.
[0078] One material from which a matrix is preferably made that
generally has at least some of the preferred properties described
above is a gel, especially a hydrogel. Suitable gels are as
described above for gel picowells of a multiwell plate of the
present invention.
[0079] In a preferred embodiment of the present invention, the
matrix further comprises an active entity. A preferred active
entity is an indicator, especially an indicator configured to
indicate a cell response to a stimulus, such as the release of a
second active entity.
[0080] According to the teachings of the present invention there is
also provided a method for handling living cells, comprising: (a)
providing an ordered array of living cells immobilized in a
non-fluid matrix, the matrix configured to maintain cell viability;
(b) contacting the living cells with a stimulus; and (c) detecting
a response of the cells to the stimulus. The method of handling
living cells of the present invention is preferably implemented
using the device of the present invention.
[0081] In an embodiment of the present invention, the matrix
further comprises an active entity. A preferred active entity is an
indicator, especially an indicator configured to indicate a cell
response to a stimulus, such as the release of a second active
entity.
[0082] In an embodiment of the present invention, part of the
detecting a response comprises contacting the matrix with an active
entity. A preferred active entity is an indicator, especially an
indicator configured to indicate a cell response to a stimulus,
such as the release of a second active entity. In some embodiments,
it is required to wait a period of time so as to allow the
contacted active entity to reach proximity with the cells, for
example by diffusion through the matrix.
[0083] In an embodiment of the present invention, detecting
comprises detecting emitted light, for example light emitted by a
cell or from an indicator, for example by fluorescence. In an
embodiment of the present invention, detecting comprises detecting
light, for example light reflected, diffracted, passing through or
passing by a cell or an indicator.
[0084] According to the teachings of the present invention there is
provided a method for producing an ordered array of living cells in
a non-fluid matrix, comprising: (a) providing a multiwell plate
provided with a plurality of wells, the multiwell plate including a
plurality of picowells at the bottom of at least one well, the
plurality of picowells including picowells; (b) placing a
suspension of a plurality of living cells in a gellable fluid in
the at least one well; (c) causing the living cells to settle into
the picowells so as to be held in respective picowells; and (d)
gelling the gellable fluid so as to make a gel cover, trapping the
living cells between the picowells and the gel cover. In an
embodiment of the present invention, the picowells are made of a
material comprising a gel.
[0085] Generally, causing the living cells to settle into the
picowells includes applying a force to the cells, typical forces
including gravitation, centrifugal forces, forces resulting from
the impact of photons on the cells (e.g., laser tweezers,
application of a non-focussed laser (see, for example, P.A.L.M.
Microlaser Technologies AG, Bernried, Germany)), or forces
resulting from a pressure wave (such as produced by an ultrasonic
transponder).
[0086] In a preferred embodiment, prior to gelling the gellable
fluid, it is ensured that each picowell holds no more than one
living cell. In another preferred embodiment, prior to gelling the
gellable fluid, it is ensured that each picowell holds no more than
a predetermined number of living cell or holds a predetermined
number of living cells.
[0087] In a preferred embodiment, the gellable fluid is chosen so
that upon gelling a transparent gel is formed. In a preferred
embodiment, the gellable fluid is chosen so that upon gelling a
hydrogel is formed.
[0088] Depending on the nature of the gellable fluid used,
preferred methods of gelling the gellable fluid include of heating
the gellable fluid, cooling the gellable fluid, irradiating the
gellable fluid, illuminating the gellable fluid, contacting the
gellable fluid with a gelling reagent and waiting a period of time
for the gellable fluid to gel. Gellable fluids suitable for use in
in implementing the method of the present invention include but are
not limited to agar gel solutions, agarose gel solutions, gelatin
solutions, low melting temperature agarose gel solutions, alginate
gel solutions, room-temperature Ca.sup.2+-induced alginate gel
solutions and polysaccharide gel solutions. Depending on the
embodiment, a gellable fluid has a water content of greater than
about 80% by weight, greater than about 92% by weight, greater than
about 95% by weight, greater than about 97% by weight and even
greater than about 98% by weight. A preferred gellable fluid is an
alginate solution where gelling the gellable fluid includes
contacting the gellable fluid with a gelling reagent, such as a
gelling reagent including Ca.sup.2+ ions. An additional preferred
gellable fluid is a low melting temperature agarose solution and
gelling the gellable fluid includes cooling the gellable fluid. In
an embodiment of the present invention, the gellable fluid further
comprises an active entity. A preferred active entity is an
indicator, especially an indicator configured to indicate a cell
response to a stimulus, such as the release of a second active
entity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0090] In the drawings:
[0091] FIG. 1 (prior art) depicts a cell-chip device of PCT patent
application IL01/00992 including a transparent carrier;
[0092] FIG. 2 (prior art) is a reproduction of a photograph of a
cell-chip device of PCT patent application IL01/00992;
[0093] FIG. 3 (prior art) is a reproduction of a photograph of a
cell-populated well-array of a carrier of a cell-chip device of PCT
patent application IL01/00992;
[0094] FIG. 4 (prior art) is a schematic depiction of a standard
commercially available 96-well plate;
[0095] FIGS. 5A-5B are reproduction of photographs of a 96-well
plate of the present invention showing wells and picowells;
[0096] FIG. 6 is a reproduction of a scanning electron micrograph
of an array of picowells of a multiwell plate of the present
invention;
[0097] FIG. 7 is a schematic depiction of a side view of picowells
of the present invention configured as enclosures;
[0098] FIG. 8 is a reproduction of a scanning electron micrograph
of the domes on a nickel template used for the production of a
plurality of picowells of the present invention;
[0099] FIGS. 9A-9F are schematic depictions of steps of a method of
the present invention for making a multiwell plate of the present
invention by contacting a template bearing negatives of wells and a
pluralities of picowells with a reversibly deformable precursor
material;
[0100] FIGS. 10A-10C are schematic depictions of steps of a method
of the present invention for making a multiwell plate of the
present invention by contacting a template bearing negatives of a
plurality of picowells with a reversibly deformable precursor
material inside a well of preexisting multiwell plate;
[0101] FIGS. 11A-11E are schematic depictions of steps of a method
of the present invention for making a multiwell plate of the
present invention by contacting a template bearing negatives of
pluralities of picowells with a reversibly deformable precursor
material followed by attachment of a separate well-wall
component;
[0102] FIGS. 12A-12D are schematic depictions of steps of a method
of the present invention for making a multiwell plate of the
present invention by producing picowells on a flat precursor plate
using photolithography followed by attachment of a separate
well-wall component;
[0103] FIGS. 13A-13C are schematic depictions of steps of a method
of the present invention for making a multiwell plate of the
present invention by producing pluralities of picowells by
photolithography inside wells of a preexisting multiwell plate;
[0104] FIGS. 14A-14C are schematic depictions of steps of a method
of the present invention for making a multiwell plate of the
present invention by attaching preexisting picowell-bearing
carriers inside wells of a preexisting multiwell plate;
[0105] FIGS. 15A-15C are schematic depictions of steps of a method
of the present invention for making a multiwell plate of the
present invention by attaching preexisting picowell-bearing
carriers to a substantially flat precursor plate followed by
attachment of a separate well-wall component;
[0106] FIG. 16 is a schematic depiction of a device of the present
invention being substantially a 3 by 3 array of living cells held
in a non-fluid matrix; and
[0107] FIG. 17 is a schematic depiction of a 96-well plate of the
present invention comprising arrays of living cells in a non-fluid
matrix.
DETAILED DESCRIPTION OF THE INVENTION
[0108] The present invention is of a mulitwell plate having a
plurality of picowells on the bottom of the wells of the plate. The
present invention is also of methods of producing a mulitwell plate
of the present invention. The present invention is also of a device
comprising an array of living cells held in a non-fluid matrix. The
present invention is also of a method of handling living cells by
providing an ordered array of living cells immobilized in a
non-fluid matrix, contacting the living cells with a stimulus; and
detecting a response to the stimulus. The present invention is also
of a method of producing an ordered array of living cells.
[0109] The principles and uses of the teachings of the present
invention may be better understood with reference to the
accompanying description, figures and examples. In the figures,
like reference numerals refer to like parts throughout.
[0110] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth herein. The invention
can be implemented with other embodiments and can be practiced or
carried out in various ways. It is also understood that the
phraseology and terminology employed herein is for descriptive
purpose and should not be regarded as limiting.
[0111] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include techniques
from the fields of biology, chemistry and engineering. Such
techniques are thoroughly explained in the literature. Unless
otherwise defined, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which the invention belongs. In addition, the
descriptions, materials, methods, and examples are illustrative
only and not intended to be limiting. Methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of the present invention. All publications, patent
applications, patents and other references mentioned are
incorporated by reference in their entirety as if fully set forth
herein. In case of conflict, the specification herein, including
definitions, will control.
[0112] As used herein, the terms "comprising" and "including" or
grammatical variants thereof are to be taken as specifying the
stated features, integers, steps or components but do not preclude
the addition of one or more additional features, integers, steps,
components or groups thereof. This term encompasses the terms
"consisting of" and "consisting essentially of".
[0113] The phrase "consisting essentially of" or grammatical
variants thereof when used herein are to be taken as specifying the
stated features, integers, steps or components but do not preclude
the addition of one or more additional features, integers, steps,
components or groups thereof but only if the additional features,
integers, steps, components or groups thereof do not materially
alter the basic and novel characteristics of the claimed
composition, device or method.
[0114] The term "method" refers to manners, means, techniques and
procedures for accomplishing a given task including, but not
limited to, those manners, means, techniques and procedures either
known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
Implementation of the methods of the present invention involves
performing or completing selected tasks or steps manually,
automatically, or a combination thereof.
[0115] Herein, the term "active entity" is understood to include
chemical, biological or pharmaceutical entities including any
natural or synthetic chemical or biological substance that
influences a cell with which the entity is in contact. Typical
active entities include but are not limited to active
pharmaceutical ingredients, antibodies, antigens, biological
materials, chemical materials, chromatogenic compounds, drugs,
enzymes, fluorescent probes, immunogenes, indicators, ligands,
nucleic acids, nutrients, peptides, physiological media, proteins,
receptors, selective toxins and toxins.
[0116] Herein, by "indicator" is meant any active entity that upon
interaction with some stimulus produces an observable effect. In
the context of the present invention, by stimulus is meant, for
example, a specific second active entity (such as a molecule)
released by a cell and by observable effect is meant, for example,
a visible effect, for example a change in color or emission of
light.
[0117] Some embodiments of the present invention include components
that are transparent or are made of a transparent material. By
"transparent" is meant that the component or material is
substantially transparent to radiation having a wavelength in at
least part of the visible light spectrum, the ultraviolet light
spectrum and/or of infrared radiation, preferably the visible light
spectrum.
[0118] It is important to note that some embodiments of the present
invention are related to embodiments of unpublished copending PCT
patent application IL04/00571 of the Applicant filed 27 Jun. 2004.
In IL04/00571 are taught picowell-bearing carriers having a variety
of innovative features. One aspect of the teachings of PCT patent
application IL04/00571 is of picowells configured to influence cell
proliferation of cells held therein. In one embodiment, carriers
having picowells of a changeable size is taught. In another
embodiment, carriers configured to delay proliferation of cells
held therein, for example by delaying or preventing cell adhesion,
are taught. In another embodiment, carriers configured so as to
allow cells to grow into or through the carrier are taught. The
above-described embodiments are preferably implemented by making
the picowells of or coating the picowells with a material with the
desired properties. In some embodiments, the inner surface of a
picowell with which a held cell makes contact is configured to have
the desired property, influence or effect. Preferred materials from
which to make carriers listed in PCT patent application IL04/00571
include polydimethylsiloxane, elastomers (such as silicon rubber),
polymerized para-xylylene molecules, polymerized derivatives of
para-xylylene molecules and gels (especially hydrogels). In some
embodiments, the inner surface of a picowell with which a held cell
makes contact is configured to have the desired property, influence
or effect.
[0119] An additional aspect of PCT patent application IL04/00571
are the teachings of a gel cover for picowell bearing components.
The gel cover is configured to prevent cells held in a picowell
from exiting the picowell due to jostling, incidental fluid flows
or during movement of the carrier.
[0120] The advantages of a picowell-bearing carrier made of a gel,
of a picowell gel-cover or a gel carrier covered with a gel cover
include, depending on the embodiment, that active entities may be
integrated into the gel, that active entities may be contacted with
the cell by diffusion through the gel, that diffusion of released
compounds is slowed down allowing identification of which cell
released a given compound, that proliferation of cells held therein
is delayed but once cells begin to proliferate, that allows
proliferation into and through the gel matrix.
[0121] As discussed hereinabove, a prior art multiwell plate is
substantially a planar device having an upper surface whereupon is
found an array of wells configured to hold a fluid containing cells
or other entities. As noted above, multiwell plates generally have
a standard footprint of ca. 8.5 cm by ca. 12.5 cm. As noted above,
the wells of a prior art multiwell plate are generally distributed
in a standard 2n by 3n rectangular packed well-array, n being an
integer. The standard multiwell plates have 6, 12, 48, 96, 384 or
even 1536 standard sized wells. The volume of the wells depends on
the number of wells and the depth thereof but is generally greater
than 5.times.10.sup.-6 liter (for a 1536 well plate). In FIG. 4 is
depicted a top view of a prior art 96-well plate 42 from the top,
comprising 96 wells 44 arranged in a 8 by 12 array.
[0122] The present invention provides an improved multiwell plate
where at the bottom surface of at least one of the wells
(preferably substantially all of the wells) are a plurality of
picowells. FIG. 5 are top views of a multiwell plate of the present
invention. With no magnification, a plate of the present invention
looks like prior art plate 42 depicted in FIG. 4. Magnification of
a single well 44 of the 96 wells reveals that at the bottom of a
well 44 is found an array 18 of hexagonally packed 20 micron
hexagonal picowells 46, FIGS. 5A and 5B. In one embodiment,
substantially the entire bottom surface of such a well comprises
picowells (as depicted in FIG. 5A). In one embodiment of the
multiwell plate of the present invention, the picowell-containing
wells are homogenous, that is all have substantially the same size
and arrangement of picowells (as depicted in FIG. 5A). In another
embodiment of the multiwell plate of the present invention, the
picowell-containing wells are heterogenous, that is there is
variation between wells, for example variation in the size of the
picowells, the arrangement of the picowells or the material from
which the picowells are made or with which the picowells are
coated.
[0123] The present invention also provides methods of making
multiwell plates of the present invention. According to one
embodiment of the method of the present invention, a preexisting
multiwell plates is converted into a multiwell plates of the
present invention. According to another embodiment of the method of
the present invention, a multiwell plates of the present invention
is fashioned in one piece, the wells and the picowells being
integrally formed components of the multiwell plate. According to
another embodiment of the method of the present invention, a
multiwell plates of the present invention is fashioned by attaching
a component or a plurality of components that is substantially the
walls defining the wells to a second component, where the second
component is substantially a plate bearing the picowells of the
multiwell plate.
Multiwell Plate of the Present Invention
[0124] As stated hereinabove, a multiwell plate of the present
invention is substantially a multiwell plate having a plurality of
wells wherein at the bottom surface of at least one well of the
plate is found a plurality of picowells. Preferably, such a plate
has a footprint of a standard multiwell plate. Preferably, the
wells of the plurality of wells of such a plate are arranged in a
manner similar or substantially identical to the arrangement of
wells of a standard multiwell plate, that is, a rectangular packing
of 6n wells arranged in a 2n by 3n array, where n is an integer
greater than 0. Preferred are the most common multiwell plate
formats, that is, 6, 24, 96, 384 and 1536 wells, 96-wells and
384-wells being most preferred. Preferably, the individual
picowells of the plurality of picowells are individually
addressable. For ease of optical study and observation, it is
preferred that the bottoms of all the picowells of a certain well
or of the entire plate be substantially coplanar: coplanarity
allows for optical observation of many cells (whether by scanning
or simultaneously using a wide-angle observation component) without
the need for time consuming and technically difficult to implement
refocusing.
[0125] The use of a multiwell plate of the present invention allows
efficient study of pluralities of living cells as individuals.
[0126] On the one hand, standard accessories available in the art
for manipulating and using multiwell plates including robotic plate
handlers, robotic fluid dispensers, multipipettes, multifilters and
the like are useable with the multiwell plates of the present
invention. Further, the format of the wells of prior art multiwell
plates has proven to be convenient for the performance of many
simultaneous experiments in the field of cellular biology, for
example, during combinatorial studies.
[0127] On the other hand, cells placed in a well of a multiwell
plate of the present invention are held in the picowells of a
respective plurality of picowells. The effect is that a plurality
of cells held in a multiwell plate of the present invention are
arranged in a rationally ordered array. The rational arrangement of
cells eases observation (especially when the bottoms of the
picowells are coplanar) and makes the cells more easily observable
as individuals (especially when the picowells are individually
addressable). Held cells are isolated from direct physical contact
with other cells, improving the quality of experimental
results.
[0128] In an embodiment of the present invention, the picowells of
the plurality of picowells of a well are juxtaposed. By juxtaposed
is meant that in an area where picowells are found, most of the
area is picowell area and little of the area is inter picowell
area. For example, in embodiments of the present invention, the
inter picowell area between two picowells is less than or equal to
0.35, 0.25, 0.15, 0.10 or even 0.06 of the sum of the areas of the
two picowells. In a preferred embodiment, the inter picowell area
is substantially nonexistent, for example when the rims of
picowells are substantially knife-edged. A picowell-array having
substantially no inter picowell area is seen in FIG. 5B. In FIG. 6,
a reproduction of a scanning electron micrograph of a
picowell-array of a multiwell plate of the present invention having
knife-edged rims is shown. One advantage of juxtaposed picowells is
that when cells are placed in a respective well, the cells settle
into picowells and do not settle onto inter picowell areas.
[0129] Further, when a plurality of juxtaposed picowells is used, a
near-tissue density planar array of cells is achieved. For example,
an array of 10-micron wide hexagonal packed knife-edged picowells
has a picowell density of about 1.5.times.10.sup.6 picowells
cm.sup.-1.
[0130] Further, for reasons of a simple loading procedure and a
high picowell density, in a preferred embodiment of the present
invention, a plurality of picowells covers substantially the entire
bottom surface of a respective well, as depicted in FIG. 5.
[0131] As the teachings of the present invention are directed to
cellular biology, it is generally preferred that the picowells be
small so as to avoid having a large number of cells held in any one
picowell. Thus, generally, the dimensions of the picowells are
generally less than about 200, 100, 50, 25 or even 10 microns. By
dimensions is meant the usual meaning of the word and is dependent
on the shape of the picowell. For example, for hexagonal or
circular picowells, the term dimension refers to diameter. For
square or triangular picowells is meant the longest dimension of
the square or triangle, respectively. The exact dimensions of
individual picowells depends on the type (and consequently size) of
cells to be studied and the types of experiments and studies that
are to be performed. Since different types of cells have different
sizes, generally a multiwell plate of the present invention has
picowells of a size to accommodate one or more cells of the type to
be studied. In some embodiments it is preferred that an individual
picowell be of a size so as to hold no more than one living cell of
a certain size. In other embodiments it is preferred that the
picowell be of a size so as to held no more than a predetermined
number of cells of a certain size (e.g., two or three cells
simultaneously).
[0132] In some embodiments of the present invention, picowells are
dimples or depressions on the bottom surface of the inside of a
well of a multiwell plate, as seen in FIG. 6. In other embodiments,
depicted in side view in FIG. 7, picowells 46 are substantially
enclosures of dimensions so that at least one cell 48 of a certain
size is containable, substantially entirely, within the enclosure,
each enclosure having an opening 50 at the surface, the opening
defined by a first cross section of a size allowing passage of cell
of the certain size 48. The exact dimensions of the individual
enclosures depends on the type (and consequently size) of cells to
be studied and the types of experiments and studies that are to be
performed. The volume of such enclosure picowells is typically less
than 1.times.10.sup.-11 liter (corresponding to the volume of a 200
micron cube), less than 1.times.10.sup.-12 liter (corresponding to
the volume of a 100 micron cube), less than 1.times.10.sup.-13
liter (corresponding to the volume of a 50 micron cube), less than
1.times.10.sup.-14 liter (corresponding to the volume of a 25
micron cube) and even less than 1.times.10.sup.-15 liter
(corresponding to the volume of a 10 micron cube). The area of the
first cross section, corresponding to the size of the opening of a
respective enclosure is typically less than about 40000
micron.sup.2 (corresponding to the area of a 200 micron square),
10000 micron.sup.2 (corresponding to the area of a 100 micron
square), 2500 micron.sup.2 (corresponding to the area of a 50
micron square), 625 micron.sup.2 (corresponding to the area of a 25
micron square) or even less than about 100 micron.sup.2
(corresponding to the area of a 10 micron square).
[0133] In embodiments of the present invention, all the picowells
of all the pluralities of picowells in all the wells of the
multiwell plate of the present invention are substantially
identical in size. In embodiments of the present invention, the
plurality of picowells in one well is substantially different from
the plurality of picowells in a second well. For example, in an
embodiment of the present invention the size of the picowells of
the plurality of picowells in one well is different from the size
of the picowells of the plurality of picowells in a second well. In
embodiments of the present invention, the plurality of picowells in
one well includes picowells of different sizes or shapes. For
example, in an embodiment of the present invention, one well
includes 10 micron picowells together with 20 micron micron
picowells.
[0134] A multiwell plate of the present invention is made of any
suitable material. Suitable materials include but are not limited
to ceramics, elastomers, epoxies, glasses, glass-ceramics, metals,
plastics, polycarbonates, polydimethylsiloxane, polyurethane,
polyethylenterephtalate glycol, polymers, polymethyl methacrylate,
polystyrene, polyvinyl chloride, rubber, silicon, silicon oxide and
silicon rubber. In an embodiment of the present invention, the
bottom surface of the wells is made of any suitable material.
Suitable materials include but are not limited to ceramics,
elastomers, epoxies, glasses, glass-ceramics, metals, polymers,
plastics, polycarbonates, polydimethylsiloxane,
polyethylenterephtalate glycol, polymethyl methacrylate,
polystyrene, polyurethane, polyvinyl chloride, rubber, silicon,
silicon oxide and silicon rubber.
[0135] In embodiments of the present invention, an entire plate of
the present invention and all components thereof are made of one
material. In other embodiments, a plate of the present invention is
made up of a number of different materials, for example, as a
plurality of layers or as a coated structure.
[0136] In an embodiment of the present invention, the walls of
wells are integrally formed with the bottom surface of the wells.
In embodiments, a multiwell plate of the present invention
comprises at least one distinct well-wall component attached to the
bottom surface. Such a distinct well-wall component is made of any
suitable material. Suitable materials include but are not limited
to ceramics, elastomers, epoxies, glasses, glass-ceramics, metals,
plastics, polycarbonates, polydimethylsiloxane,
polyethylenterephtalate glycol, polymers, polyurethane, polymethyl
methacrylate, polystyrene, polyvinyl chloride, rubber, silicon,
silicon oxide and silicon rubber.
[0137] In embodiments of the present invention, a plurality of
picowells is integrally formed with the bottom surface of a
respective well.
[0138] In embodiments of the present invention, a multiwell plate
of the present invention comprises at least one distinct
picowell-bearing component bearing a plurality of picowells, the
component resting in or attached to the bottom surface of a
respective well. A suitable distinct picowell-bearing component is
a carrier comprising a plurality of picowells disposed on a
surface, such as a carrier described in PCT patent application
IL01/00992 or in unpublished copending PCT patent application
IL04/00571 of the Applicant filed 27 Jun. 2004. Picowell-bearing
components are made of any suitable material, including reversibly
deformable materials and irreversibly deformable materials.
Suitable materials include but are not limited to gels, hydrogels,
waxes, hydrocarbon waxes, crystalline waxes, paraffins, ceramics,
elastomers, epoxies, glasses, glass-ceramics, metals, plastics,
polycarbonates, polydimethylsiloxane, polyethylenterephtalate
glycol, polymers, polymethyl methacrylate, polystyrene,
polyurethane, polyvinyl chloride, rubber, silicon, silicon oxide
and silicon rubber.
[0139] In an embodiment of the multiwell plate of the present
invention, a picowell-bearing component comprises a gel, preferably
a transparent gel, preferably a hydrogel. Gel picowell-bearing
components are discussed in detail in PCT patent application
IL04/00571. As will be discussed in detail hereinfurther, in
general a gel picowell-bearing component of the present invention
is advantageously produced by placing a gellable fluid in a well of
an existing multiwell plate, contacting the gel with a template
including, amongst others, negatives of the picowells, and then
gelled. Gels suitable for use in making a picowell-bearing
component of a plate of the present invention include but are not
limited to agar gels, agarose gels, gelatins, low melting
temperature agarose gels, alginate gels, room-temperature
Ca.sup.2+-induced alginate gels and polysaccharide gels. Depending
on the embodiment, a suitable gel has a water content of greater
than about 80% by weight, greater than about 92% by weight, greater
than about 95% by weight, greater than about 97% by weight and even
greater than about 98% by weight. Two exceptionally preferred types
of hydrogels are alginates and low melting temperature
agaroses.
[0140] Alginates are biologically compatible polysaccharide
proteins that are fluid at low calcium ion concentrations (e.g.,
[Ca.sup.2+]<1 .mu.M) but gel upon exposure to higher
concentrations of calcium ions (e.g., [Ca.sup.2+]=20 mM). An
exceptionally suitable alginate for implementing the teachings of
the present invention is sodium alginate and may be purchased, for
example, from Pronova Biopolymers (Drammen, Norway) as Protanal
LF120 1% in water or Protanal LF200 1% in water.
[0141] Low melting temperature agaroses are biologically compatible
gels that before gelling are fluid at temperatures that do not harm
living cells (e.g., 20.degree. C.), gel at low temperatures that do
not harm living cells (e.g., 4.degree. C.) and remain stable until
well-above temperatures used for studying living cells (40.degree.
C.). An exceptionally suitable agarose for implementing the
teachings of the present invention that may be purchased, for
example, from Cambrex Bio Science Rockland Inc. (Rockland, Me.,
USA) is HGS-LMP Agarose (catalogue nr. 50221).
[0142] In an embodiment, the gel includes an active entity.
Suitable active entities include, but are not limited to
antibodies, antigens, biological materials, chemical materials,
chromatogenic compounds, drugs, enzymes, fluorescent probes,
immunogenes, indicators, ligands, nucleic acids, nutrients,
peptides, physiological media, proteins, receptors, selective
toxins and toxins.
[0143] In an embodiment of the present invention, picowells have a
bottom surface made of a first material and borders, such as the
borders delineating the picowells, made of a second material, the
second material being substantially different from the first
material. In an embodiment of the present invention the first
material is substantially the material from which the bottom of the
well is made, for example when the bottom surface of the picowell
is substantially the bottom surface of the well. In an embodiment
of the present invention, the second material is a fixed
photoresist material. As is detailed hereinbelow, such a picowell
structure is achieved by fixing a photoresist material applied to a
precursor plate. An advantage of such like plates is that features
such as picowells having flat bottom surfaces are easily made.
[0144] In an embodiment of the multiwell plate of the present
invention, picowells are configured with an inside surface
configured to delay proliferation of cells held therein, for
example by delaying adhesion of living cells thereto. Picowells
configured to delay proliferation of living cells held therein are
discussed in detail in PCT patent application IL04/00571. In an
embodiment of the plate of the present invention, the inside of a
picowell comprises a material that delays adhesion of living cells
thereto, that is the picowell is substantially fashioned from the
adhesion-delaying material or the inside of the picowell is coated
with the adhesion-delaying material. A suitable material to coat
the inside of a picowell or from which to make a picowell comprises
polydimethylsiloxane, is substantially polydimethylsiloxane or is
substantially pure polydimethylsiloxane. Suitable
polydimethylsiloxane resins for coating picowells or to make
picowells are commercially available and can be purchased, amongst
others, under the trade names RTV615 PDMS (GE Silicones, Wilton,
Conn., USA) and Sylgard 184 PDMS (Dow Corning Corporation, Midland,
Mich., USA).
[0145] In an embodiment of the multiwell plate of the present
invention, bottom surfaces of the picowells comprise a material
having an index of refraction similar to that of water, that is an
index of refraction of less than about 1.4, less than about 1.38,
less than about 1.36, less than about 1.35, less than about 1.34 or
substantially equal to that of water. Picowells having indicia of
refraction similar to that of water are discussed in detail in PCT
patent application IL04/00571. An advantage of such picowells is
that observation of cells is simplified as the picowell walls are
substantially invisible and there is little, if any, scattering,
reflection and diffraction of light, that otherwise interferes with
optical study of held cells, for example, during morphological
studies using a microscope.
[0146] In an embodiment of the present invention, the plurality of
picowells comprises picowells having an inner surface coated with a
layer of a material. Suitable materials for coating an inner
surface of a picowell of a plate of the present invention include
but are not limited to gels, hydrogels, polydimethylsiloxane,
elastomers, polymerized para-xylylene molecules, polymerized
derivatives of para-xylylene molecules, rubber and silicon rubber.
Picowells having coated inner surfaces are discussed in detail in
PCT patent application IL04/00571.
[0147] In an embodiment of the present invention, a plate of the
present invention further comprises a gel cover covering a
plurality of the picowells, the cover made of a gel. Suitable gels
are as described herein. Gel picowell covers are discussed in
detail in PCT patent application IL04/00571.
[0148] In an embodiment of the present invention, a multiwell plate
of the present invention further comprises at least one additional
feature functionally associated with the plurality of picowells,
especially a microfluidic feature. Suitable microfluidic features
include but are not limited to channels, coupling elements, drains,
fluid channels, fluid reservoirs, input ports, membranes,
microreactors, microvalves, output ports, passages, plumbing
routes, protruberances, pumps, transport channels and valves. Other
suitable features include but are not limited to light sources,
magnetizable elements, optical components, optical fibers, optical
filters, protuberances, fiducial points and walls. Such an
embodiment can be considered to be a multiwell plate of the present
invention that holds a carriers such as described in PCT patent
application IL01/00992 or in unpublished copending PCT patent
application IL04/00571. Such an embodiment allows performance of
many and varied experiments to study living cells, as described in
the above references.
[0149] In an embodiment of the of the present invention, a
multiwell plate further comprises a cover slip, the cover slip and
a plurality of picowells in a well configured so as to allow the
cover slip to rest above the plurality of picowells substantially
in parallel to the bottom surface of the respective well. Such a
cover slip can include microelectrodes to assist in manipulation of
cells held in picowells, can be used in conjunction with other
features so as to provide a microfluidics system for the picowells,
or for other reasons as discussed in PCT patent application
IL01/00992.
Methods of Manufacture of a Multiwell Plate of the Present
Invention
[0150] A multiwell plate of the present invention is produced using
any suitable method known in the art. Suitable methods include
methods that employ one or more techniques including but not
limited to casting, embossing, etching, free-form manufacture,
injection-molding, microetching, micromachining, microplating,
molding, spin coating, lithography or photo-lithography.
[0151] The preferred methods of producing a multiwell plate of the
present invention are the methods of the present invention.
[0152] A first method of the present invention for making a
multiwell plate of the present invention is substantially by
contacting a precursor material with a template, the template
having a negative of some of the features of the plate (especially
the picowells) thus creating the features in the precursor
material. The features are subsequently fixed in the precursor
material making an incipient plate. After any further processing of
the incipient plate required (which may be limited to simply
separating the template from the incipient plate), the multiwell
plate of the present invention is fashioned.
[0153] Depending on the precursor material, fixing includes, but is
not limited to, methods such as heating the precursor material,
cooling the precursor material, curing the precursor material,
polymerizing the precursor material, cross-linking the precursor
material, irradiating the precursor material, illuminating the
precursor material, gelling the precursor material, exposing the
precursor material to a fixative and waiting a period of time. By
fixative is meant an agent that causes the precursor material to
change to the fixed state and is used herein as a general term for
such materials as fixatives, hardeners,
polymerization/crosslinking/curing initiators, catalysts and
agents. It is important to note that in some cases a precursor
material is produced by mixing two or more components which
thereafter change to a fixed state, for example, by simply waiting
a period of time.
[0154] In one preferred embodiment of the present invention, the
precursor material is a irreversibly deformable precursor material.
Herein by irreversibly deformable precursor material is meant a
material that does not recover a shape after deformation and so
there is usually no need for a separate action to fix the features
in the precursor material beyond separating the produced multiwell
plate from the template. In such cases, the precursor material does
not substantially chemically change subsequent to contact with the
template. Examples of suitable irreversibly deformable precursor
materials include waxes, paraffins, plastics, polymers and the
like. In such an embodiment, a preferred template is a stamp, and
the contacting of the template with the precursor material is
substantially stamping the features of the multiwell plate into the
precursor material, preferably under controlled thermal
conditions.
[0155] In another preferred embodiment of the present invention,
the precursor material is a reversibly deformable precursor
material. Herein by reversibly deformable precursor material is
meant a material that is capable of recovering shape after
deformation and includes gellable fluids, polymerizable materials,
powders, fluids and thermoplastic materials.
[0156] In a preferred embodiment, the reversibly deformable
precursor material is a thermoplastic material at a pliable
temperature. Subsequent to the contacting of the template but
before the contact is finished, the thermoplastic material is
cooled, thus fixing the desired features in the incipient multiwell
plate.
[0157] In another preferred embodiment, the reversibly deformable
precursor material is a polymerizable material (e.g., a monomer
solution, a crosslinkable polymer, a vulcanizable polymers, a
polymerizable fluids or a thermosetting resin). Subsequent to the
contacting of the template but before the contact is finished, the
polymerizable material is polymerized, thus fixing the desired
features in the incipient multiwell plate. In such cases, the
precursor material and the material from which the multiwell plate
is made are chemically dissimilar (for example, have the
relationship of monomer to polymer).
[0158] One preferred polymerizable precursor material is a
non-cured polydimethylsiloxane precursor mixture. A mixture of two
polydimethylsiloxane components (the prepolymer and curing agent)
are mixed together in the desired ratio (preferably about 10:1, but
ratios between about 5:1 and about 20:1 are generally suitable) to
give a polydimethylsiloxane precursor mixture, the mixture degassed
and contacted with the template. The features are fixed by the
curing of the mixture. Curing of polydimethylsiloxane precursor
generally takes place at room temperature for about 24 hours and,
when desired, is accelerated by heating. For example it has been
found that multiwell plates of the present invention made of
polydimethylsiloxane are ready for further processing within 2
hours when cured at 60.degree. C. or within 15 minutes when cured
at 150.degree. C. A detailed review of methods for the production
of micronic features such as picowells from polydimethylsiloxane
suitable for implementing the teachings of the present invention
are known in the art and discussed, for example, in Ng et al.,
Electrophoresis 2002, 23, 3461-3473 and Duffy et al., Anal. Chem.
1998, 70, 4974-4984.
[0159] Another preferred polymerizable precursor material is
urethane that is polymerized to yield polyurethane.
[0160] Another preferred reversibly deformable precursor material
is a gellable fluid. After the gellable fluid is brought in contact
with the template, the features are fixed by gelling the gellable
fluid to yield a gel. Most preferred are gellable fluids that
produce a hydrogel.
[0161] Gellable fluids known in the art include fluids that gel
upon heating, fluids that gel upon cooling, fluids that gel upon
irradiation or illumination, fluids that gel as a result of contact
with a gelling reagent and fluids that gel after a period of time.
Preferred gellable fluids for implementing the teachings of the
present invention include solutions of agars, agaroses, gelatins,
low melting temperature agaroses, alginates, proteins, protein
polysaccharides, Ca.sup.2+-inducable alginates (especially those
that gel at room temperature) and polysaccharides.
[0162] One preferred gellable fluid is a low-melting temperature
agarose solution. Such a solution is fluid at temperatures that do
not harm living cells (e.g., 20.degree. C.) and gel at low
temperatures that do not harm living cells (e.g., 4.degree. C.). An
exceptionally suitable agarose for implementing the teachings of
the present invention that may be purchased, for example, from
Cambrex Bio Science Rockland Inc. (Rockland, Me., USA) is HGS-LMP
Agarose 0.5% in PBS (catalogue nr. 50221).
[0163] Another preferred gellable fluid is an alginate solution
which gels upon contact with a gelling reagent, the preferred
gelling reagent being a solution having a Ca.sup.2+ ion
concentration of greater than about 1.times.10.sup.-6 M. An
exceptionally useful gelling agent is a 20.times.10.sup.-3 M
calcium gluconate solution. Suitable alginate solutions can be
purchased from Pronova Biopolymers (Drammen, Norway) and include,
for example, Protanal LF120 1% in water and Protanal LF200 1% in
water.
[0164] The template having a negative of the features is, for
example, a stamp or a mold, and is generally made of any suitable
material that is more rigid than a respective precursor material.
Suitable materials include but are not limited to reversibly
deformable materials, irreversibly deformable materials, ceramics,
epoxies, glasses, glass-ceramics, metals, plastics, polycarbonates,
polydimethylsiloxane, polyethylenterephtalate glycol, polymers,
polymethyl methacrylate, paraffins, polystyrene, polyurethanes,
polyvinyl chloride, silicon, silicon oxide, silicon rubbers and
wax.
[0165] The template is made, for example, using methods with which
one skilled in the art is acquainted such as casting, embossing,
etching, free-form manufacture, injection-molding, microetching,
micromachining, microplating, molding, lithography or
photo-lithography.
[0166] In FIG. 8, is shown a reproduction of a scanning electron
micrograph of the domes on a nickel stamp used as a template for
the production of a multiwell plate of the present invention. Seen
is an array of hexagonally-packed domes that are the negative of a
hexagonal array of knife-edged picowells, such as seen in FIG. 6.
The diameter of the domes at the intersection with the nickel
surface is approximately 20 microns.
[0167] In some embodiments, other features created in the precursor
material by the contact of the template include features such as
drains, channels, coupling elements, drains, fiducial points, fluid
channels, fluid reservoirs, input ports, microreactors,
microvalves, passages, optical components, optical filters, output
ports, plumbing routes, protruberances, protruberances for
supporting a cover slip, pumps, transport channels, valves, walls
and partial walls.
[0168] In some embodiments of the present invention, the wells of a
multiwell plate of the present invention are made by contacting a
precursor material with a template. Steps for producing a six-well
plate of the present invention 52 according to a method of the
present invention are schematically depicted in FIG. 9. In FIG. 9A,
a fluid and therefore reversibly deformable precursor material 54
is provided. Precursor material 54 is a molten thermoplastic
material. In FIG. 9B, a template 56 substantially a nickel stamp
having features that are negatives of features of plate 52
including wells and picowells is provided. In FIG. 9C, template 56
is brought in contact with precursor material 54 so as to form the
features of plate 52 in precursor material 54. In FIG. 9D, the
features of plate 52 are fixed in precursor material 54 by cooling
so as to make incipient plate 58. After sufficient cooling,
template 56 is separated from incipient plate 58, FIG. 9E.
Incipient plate 58 undergoes whatever further processing is
necessary to ultimately yield plate 52 of the present invention,
having pluralities of picowells 18 in each one of six wells 44,
FIG. 9F.
[0169] In a preferred embodiment, a multiwell plate of the present
invention is made by making picowells (and other desired features)
as described above inside the wells of a preexisting multiwell
plate. Suitable multiwell plates include but are not limited to
plates made of reversibly deformable materials, irreversibly
deformable materials, ceramics, epoxies, glasses, glass-ceramics,
metals, plastics, polycarbonates, polydimethylsiloxane,
polyethylenterephtalate glycol, polymers, polymethyl methacrylate,
polystyrene, polyurethanes, polyvinyl chloride, silicon, silicon
oxide and silicon rubbers. In such a case, the precursor material
is placed into each desired well of the preexisting multiwell
plate. A template is then placed inside the well so as to make
contact with the precursor material and the precursor material is
fixed as described above. Such an embodiment has the advantage that
a commercially available multiwell plate of any format (e.g., 6,
24, 96, 384 and 1536 wells) and of virtually any material can be
converted into a multiwell plate of the present invention. In such
an embodiment a template can be made and used for fixing picowells
and other features in any number of wells including for each well
separately or for all wells simultaneously. In this way, a single
multiwell plate of the present invention having different features
(e.g., different sized picowells) in different wells is easily
made.
[0170] Steps for producing a six-well plate of the present
invention 52 according to a method of the present invention are
schematically depicted in FIG. 10. In FIG. 10A, a fluid and
therefore reversibly deformable precursor material 54 is placed in
a preexisting 6-well plate 60 having six wells 44. Precursor
material 54 is a non-cured polydimethylsiloxane precursor mixture
(comprising a mixture of a prepolymer and a curing agent). In FIG.
10B, a template 56 substantially a nickel stamp having features
that are negatives of features of plate 52 found in wells 44 such
as picowells and having a size and shape to precisely fit in a well
44 is contacted with precursor material 54 in each one of wells 44
sequentially so as to form the features of plate 52 in precursor
material 54 in each one of wells 44 sequentially. Template 56 is
maintained in contact with precursor material 54 in a given well 44
for so long as required for the desired features to be fixed in
precursor material 54 by solidification to be polydimethylsiloxane.
In FIG. 10B it is seen that at the bottom surfaces of each one of
three wells 44a are found a plurality of nanowells 18 while at the
bottom of each one of three wells 44b is found non-fixed precursor
material 54. Incipient plate 58 undergoes whatever further
processing is necessary to ultimately yield plate 52 of the present
invention, having pluralities of picowells 18 in each one of six
wells 44, FIG. 10C.
[0171] In another preferred embodiment, the template includes the
negative of the desired features such as picowells but not of the
wells. The template is contacted with the precursor material so as
to form a substantially planar incipient plate having the features
the negatives of which are found on the template. Subsequently, a
grid-like component, being substantially the walls of the wells of
the multiwell plate of the present invention, is attached using an
appropriate method, for example, adhesives (for example, light
curable adhesives, such as light curing adhesive 3051 or 3341
manufactured by Henkel Loctite Deutschland GmbH, Munchen, Germany)
or surface treatments such as anodic bonding, fusion bonding or
plasma treatment such as plasma discharge (exceptionally suitable
for attaching polydimethylsiloxane, see Duffy et al., Anal. Chem.
1998, 70, 4974-4984).
[0172] Steps for producing a six-well plate of the present
invention 52 according to a method of the present invention are
schematically depicted in FIG. 11. In FIG. 11A, a fluid and
therefore reversibly deformable precursor material 54 is provided.
Precursor material 54 is a non-cured polydimethylsiloxane precursor
mixture (comprising a mixture of a prepolymer and a curing agent).
In FIG. 11A, a template 56 substantially a nickel stamp having
features that are negatives of features of plate 52 such as
picowells, but not wells is also provided. In FIG. 11B, template 56
is brought in contact with precursor material 54 so as to form the
features of plate 52 in precursor material 54. Template 56 is
maintained in contact with precursor material 54 for so long as
required for the desired features to be fixed in precursor material
54 by solidification to be polydimethylsiloxane and thus to produce
a substantially planar incipient plate 58 having, amongst other
features, six pluralities of nanowells 18, FIG. 11C. In FIG. 11D, a
grid-like component 64, being substantially the walls of wells 44
of plate 52 of the present invention is provided. Attachment of
grid-like component 64 to substantially planar incipient plate 62
using adhesive (e.g., light curing adhesive 3051 manufactured by
Henkel Loctite Deutschland GmbH, Munchen, Germany) and whatever
further processing is necessary ultimately yields plate 52 of the
present invention, having pluralities of picowells 18 in each one
of six wells 44, FIG. 11E.
[0173] Another preferred method of making a multiwell plate of the
present invention includes photolithography of a photoresist
material placed on a substrate, a commercially available process
(for example, from Micro Resist Technology GmbH, Berlin, Germany)
with which one skilled in the art is well-acquainted.
[0174] In brief, a high aspect ratio photoresist material (e.g.,
SU-8 thick photoresist fluid, MicroChem Corporation, Newton Mass.,
USA) is placed on a precursor plate as a uniformly thick film. A
preferred method of achieving a uniformly thin film of a
photoresist fluid on a precursor plate is by spin coating, that is,
the photoresist fluid is placed on a surface of the precursor plate
and the precursor plate is rotated about an axis that is
perpendicular to the surface of the substrate on which the
photoresist fluid was placed. As a result of the rotation the
photoresist fluid forms a uniformly thick film on the precursor
plate, typically between about 5 microns and about 20 microns
thick. Once a film of uniform thickness of photoresist material is
achieved, the photoresist material is illuminated through a mask,
the mask being substantially a template or master of the features
which are desired to be fixed in the photoresist material including
the desired picowells. Developing of the precursor with the
selectively fixed film removes the non-fixed areas of the film. In
such a way features of a multiwell plate of the present invention
are made up of a fixed photoresist layer resting on a precursor
plate where the features of the multiwell plate are carved into the
photoresist layer and the bottom of the features (such as
picowells) is the surface of the precursor plate. Using a
photolithography method, picowells and other features are easily
produced, including features having a flat-bottom surface.
[0175] It is important to note that in addition to picowells, any
suitable feature known in the art and discussed herein for
multiwell plates of the present invention or for picowell-bearing
carriers described in PCT patent application IL 01/00992 or PCT
patent application IL 04/00571 can also be added using the
photoresist method. Such features include but are not limited to
channels, coupling elements, drains, fluid channels, fluid
reservoirs, input ports, microreactors, microvalves, passages,
output ports, plumbing routes, protruberances, pumps, transport
channels, valves, walls and fiducial points.
[0176] The material from which the precursor plate is made can be
any suitable material. Suitable materials include but are not
limited to ceramics, epoxies, glasses, glass-ceramics, metals,
plastics, polycarbonates, polydimethylsiloxane, polymers,
polyethylenterephtalate glycol, polymethyl methacrylate,
polystyrene, polyurethanes, polyvinyl chloride, silicon and silicon
oxide.
[0177] Steps for producing a six-well plate of the present
invention 52 according to a method of the present invention are
schematically depicted in FIG. 12. In FIG. 12A, a flat precursor
plate 66 is provided. In FIG. 12B, the upper surface of flat
precursor plate 66 is coated with a uniformly thin film of a
precursor material 54, precursor material 54 being a photoresist
fluid (e.g., SU-8 thick photoresist fluid, MicroChem Corporation,
Newton Mass., USA). In FIG. 12C, precursor material 54 is
illuminated through mask 68 using light source 70 so that features
such as picowells are fixed in precursor material 54. After the
features are fixed, incipient plate 58 is developed so as to remove
non fixed photoresist material and undergoes any further processing
necessary including attachment of a grid-like component 64 to
ultimately yield plate 52 of the present invention, having
pluralities of picowells 18 in each one of six wells 44, FIG.
12D.
[0178] In a preferred embodiment, the precursor plate comprises a
multiwell plate. In such a case, the photoresist material
(preferably a photoresist fluid) is placed into each desired well
of the precursor plate and the photoresist material fixed by
illumination as described above. Such an embodiment has the
advantage that a commercially available multiwell plate of any
format (e.g., 6, 24, 96, 384 and 1536 wells) and of virtually any
material can be converted into a multiwell plate of the present
invention. In such an embodiment a mask can be made and used for
fixing picowells and other features in any number of wells
including for each well separately or for all wells simultaneously.
In this way, a single multiwell plate of the present invention
having different features (e.g., different sized picowells) in
different wells is easily made.
[0179] Steps for producing a six-well plate of the present
invention 52 according to a method of the present invention are
schematically depicted in FIG. 13. In FIG. 13A, a precursor
material 54 is placed in an existing six-well plate 60 having six
wells 44. Precursor material 54 is a photoresist fluid (e.g., SU-8
thick photoresist fluid, MicroChem Corporation, Newton Mass., USA).
In FIG. 13B, a probe 72 tipped with a mask 74 and provided with a
light source 70 having a size and shape to precisely fit in a well
44 is brought in proximity with precursor material 54 in each one
of wells 44 sequentially. During the time that mask 74 is in
proximity with precursor material 54, light source 70 is activated
so that features such as picowells are fixed in precursor material
54 in a respective well 44. In FIG. 13B it is seen that at the
bottom surfaces of each one of three wells 44a are found a fixed
plurality of nanowells 18 while at the bottom of each one of three
wells 44b is found non-fixed precursor material 54. After features
are fixed in all desired wells 44, incipient plate 58 is developed
so as to remove non fixed photoresist material and undergoes any
further processing necessary. Ultimately a plate 52 of the present
invention is formed having pluralities of picowells 18 in each one
of six wells 44, FIG. 13C.
[0180] A preferred method for producing a multiwell plate of the
present invention is by producing a substantially planar incipient
plate where substantially the entire upper surface is provided with
an array of picowells, whether by contact with a temple, by
photoresist or other methods. Subsequently a well-wall component or
plurality of components is attached to the upper surface. The
well-wall component thereby defines the plurality of wells and the
upper surface of the precursor plate is substantially the bottom
surface of the wells whereupon the picowells are found.
[0181] Another preferred method of making a multiwell plate of the
present invention comprises attaching one or more picowell-bearing
components to a precursor plate using an appropriate method, for
example, using an adhesive or a surface treatment such as a plasma
treatment, for example as described above. A preferred
picowell-bearing component is a carrier comprising a plurality of
picowells disposed on a surface. Preferred carriers include those
described in PCT patent application IL 01/00992 or PCT patent
application IL 04/00571.
[0182] In a preferred embodiment, the precursor plate comprises a
multiwell plate. In such a case, one or more picowell-bearing
components are placed into one or more wells of the precursor plate
and attached using a suitable method. Such an embodiment has the
advantage that a commercially available multiwell plate of any
format (e.g., 6, 24, 96, 384 and 1536 wells) and of virtually any
material can be converted into a multiwell plate of the present
invention. In such a way many picowell-bearing components of
different types are prefabricated, for example by mass production
and placed as desired in as many wells of the precursor plate as
desired. In this way, a single multiwell plate of the present
invention having different features (e.g., different sized
picowells) in different wells is easily made.
[0183] Steps for producing a six-well plate of the present
invention 52 according to a method of the present invention are
schematically depicted in FIG. 14. In FIG. 14A, an adhesive 76
(e.g., light curing adhesive 3051 manufactured by Henkel Loctite
Deutschland GmbH, Munchen, Germany) is placed in wells 44 of an
existing six-well plate 60 having six wells 44. In FIG. 14B,
carriers 26, such as carrier 26 depicted in FIG. 2, are placed in
each one of six wells 44 and illuminated with light source 70.
Adhesive 76 is cured by exposure to light produced by light source
70, attaching each one of six carriers 26 inside a respective well
44, producing a plate 52 of the present invention, having
pluralities of picowells 18 in each one of six wells 44, FIG.
14C.
[0184] In another preferred embodiment, the precursor plate
comprises a substantially planar plate, preferably having the same
footprint of a multiwell plate (ca. 8.5 cm by ca. 12.5 cm). The
picowell-bearing components are placed in appropriate locations on
the precursor plate corresponding to the locations of one or more
wells of the ultimately made multiwell plate and attached using a
suitable method. Subsequently, a grid-like component, being
substantially the walls of the wells of the multiwell plate of the
present invention, is attached using an appropriate method, for
example, using an adhesive or a surface treatment such as a plasma
treatment.
[0185] Steps for producing a six-well plate of the present
invention 52 according to a method of the present invention are
schematically depicted in FIG. 15. In FIG. 15A, a flat precursor
plate 66 is provided. In FIG. 15B, carriers 26, such as carrier 26
depicted in FIG. 2, are attached to flat precursor plate 66, for
example using an adhesive. Attachment of grid-like component 64 to
incipient plate 58 and whatever further processing is necessary
ultimately yields plate 52 of the present invention, having
pluralities of picowells 18 in each one of six wells 44, FIG.
15C.
[0186] It is important to note that in embodiments of the method of
making a multiwell plate of the present invention by placing (and
optionally attaching) preformed picowell-bearing components to a
precursor plate, it is often advantageous that a given
picowell-bearing component have dimensions similar or substantially
identical to that of a well in which the picowell-bearing component
is attached. Such dimensions allow more exact placement of the
picowell-bearing component in the well.
[0187] Some embodiments of the multiwell plate of the present
invention comprise picowells where the inside surface of the
picowells (with which held cells potentially make physical contact)
is coated with a layer of some desired coating material, for
example a coating material that influences the proliferation of
living cells as described in PCT patent application IL04/00571.
[0188] One skilled in the art is acquainted with many ways and many
coating materials with which to coat an inside surfaces of
picowells of a multiwell plate of the present invention.
[0189] One preferred method of coating inside surfaces of picowells
of a multiwell plate of the present invention, applicable to
virtually any multiwell plate produced by virtually any method,
comprises contacting a precursor fluid with the inside surface of
the picowells and subsequently solidifying the precursor fluid,
forming the layer of the coating material. Depending on the nature
of the precursor fluid, solidifying is performed by any number of
methods including but not limited to heating the precursor fluid,
cooling the precursor fluid, polymerizing the precursor fluid,
cross-linking the precursor fluid, curing the precursor fluid,
irradiating the precursor fluid, illuminating the precursor fluid,
gelling the precursor fluid, exposing the precursor fluid to a
fixative or waiting a period of time.
[0190] One preferred method of coating the inside surfaces of
picowells of a multiwell plate of the present invention, applicable
to virtually any multiwell plate produced by virtually any method,
is by vapor deposition. Vapor deposition involves the deposition of
materials such as molecules or atoms onto a surface at low
pressures and is characterized by the production of evenly thin
coatings on a surface, such as the inner surface of a picowell of a
multiwell plate of the present invention.
[0191] In one embodiment of vapor deposition to the inside surfaces
of picowells of a multiwell plate of the present invention, the
atoms or molecules that make up the coating material are deposited.
In another embodiment of vapor deposition, the atoms or molecules
that comprise a precursor of the coating material are deposited on
the inside surfaces of the picowells, followed by solidifying the
coating precursor material thereby forming the layer of coating
material. Solidifying of the coating precursor material to form the
layer of coating material is performed by any number of methods
including but not limited to heating the coating precursor
material, cooling the coating precursor material, polymerizing the
coating precursor material, cross-linking the coating precursor
material, curing the coating precursor material, irradiating the
coating precursor material, illuminating the coating precursor
material, gelling the coating precursor material, exposing the
coating precursor material to a fixative and waiting a period of
time.
[0192] A preferred coating material for coating the inside surfaces
of picowells of a multiwell plate of the present invention is made
of polymerized para-xylylene molecules (or derivatives thereof,
specifically where one or more hydrogens, especially aromatic
hydrogens of either or both aromatic rings are substituted)
deposited by vapor deposition, a coating commercially known as
Parylene.RTM. (available for example from V&P Scientific, Inc.,
San Diego, Calif., USA). Parylene.RTM. is preferred not only for
cell proliferation influencing properties but also for the fact
that Parylene.RTM. coatings are bacteria resistant, fungus
resistant, transparent, have a low permeability, acid and base
resistant, uniform, thin (typically 0.1-1 micron) and without voids
even when a coated surface includes configurations with sharp
edges, points, flat surfaces, crevices or exposed internal
surfaces.
[0193] The teachings of the present invention provide the
possibility of providing a device useful in the field of cellular
biology. A device of the present invention comprises an array of
living cells held in a non-fluid matrix, the matrix configured to
maintain cell viability. Generally, the matrix of a device of the
present is configured to maintain cell viability. To maintain cell
viability, a matrix is generally non-cytotoxic and allows transport
of molecules necessary for cell survival and metabolism, such as
nutrients, gases, ions and waste to and from a living cell held
therein. A suitable matrix is a matrix comprising a gel, preferably
a hydrogel. Preferably, the living cells are physically held in
pockets in the matrix, especially free-floating in a physiological
fluid in the pockets. To reduce any influence on the reactions of
the cells, there is preferably substantially no bond (e.g.,
chemical bond) between the living cells and the matrix.
[0194] In FIG. 16 is depicted a device of the present invention 72,
being substantially nine living cells 74 floating inside pockets 76
inside a hydrogel matrix 78.
[0195] In a preferred embodiment, to simplify use of the device,
the array is substantially planar having an upper surface and a
lower surface. In a preferred embodiment of the present invention,
by substantially planar is meant that substantially all living
cells of the array are arranged in a single unique plane. In
another preferred embodiment of the present invention, by
substantially planar is meant that substantially all the living
cells are arranged in two or more planes.
[0196] To ease observation of the cells or detection of signals
associated with the cells, in a preferred embodiment of the device
of the present invention, one or both of the two surfaces of the
device is transparent to at least one wavelength of light,
including a wavelength of light of the ultraviolet spectrum, the
visible spectrum or the infrared spectrum. Further, to ease
observation of the cells or detection of signals associated with
the cells, in a preferred embodiment of the present invention, the
matrix comprises a material having an index of refraction
substantially similar to that of water. By substantially similar is
meant an index of refraction of less than about 1.4, less than
about 1.38, less than about 1.36, less than about 1.35 or even less
than about 1.34.
[0197] In a preferred embodiment of the present invention, the
matrix is configured to substantially delay proliferation of living
cells held therein. Configuration of a matrix so as to
substantially delay proliferation of the living cells is taught in
PCT IL04/00571. A preferred method of configuring a matrix to
substantially delay proliferation is to make at least part of the
matrix from a material that has proliferation delaying properties.
A preferred such material is a gel, especially a hydrogel.
[0198] In a preferred embodiment of the present invention, the
matrix comprises an active entity, especially an indicator. By
indicator is meant an active entity configured to indicate a cell
response to a stimulus, for example a molecule that is
chromatogenic or fluorogenic upon exposure to some compound
released by a given living cell held in the device upon exposure to
some stimulus.
[0199] The unique characteristics of a device of the present
invention are better understood by comparing the device to living
tissue on the one hand and prior art arrays of living cells on the
other hand.
[0200] As is known to one skilled in the art, living tissue can be
considered to comprise living cells held within a matrix. Further
living tissue can be maintained living for an extended period of
time. It is therefore known in the art to use devices incorporating
living tissues in a device for assaying cell response to stimuli.
However, in living tissue the cells are not in an array; the cells
are not distinct from each other making identification of
individual cell response difficult if not impossible especially
under high throughput conditions; cells are not individually
addressable so a cell of interest must be maintained under
continuous observation; cells are not coplanar making visual study
time-consuming due to the need for refocusing; cells are in contact
with each other so that one cell may influence other neighboring
cells; living tissue cannot be engineered as desired to hold
specific different cells in a desired spatial relationship to each
other; cells in living tissue proliferate, meaning that the
properties of a device including living tissue are not well-defined
and change over time. Depending on the embodiment, a device of the
present invention provides overcomes some or all of these
disadvantages.
[0201] As discussed in the introduction, prior art arrays of cells
all have a number of critical disadvantages. In some prior art
arrays, the cells are bound to some object, whether by native
adhesion or by some non-native chemical bond or attraction. Binding
a cell necessarily compromises the response of cell to stimuli. In
other prior art cell arrays, there is nothing keeping cells from
moving from a designated location so that there is no way to ensure
that array integrity is maintained. This is exceptionally
significant when cell apoptosis or other death processes occurs or
when the cell array is moved. The device of the present invention
provides, in contrast to prior art devices, a cell array that is
robust during any cell process including cell death and during
movement of the device itself.
[0202] The device of the present invention is exceptionally useful
for implementing certain manipulations of living cells. For
example, a device of the present invention, being substantially an
ordered array of selected cells, is made in a first location such
as a laboratory. The device is transported to a remote location,
for example to a second laboratory, in a space vehicle or to the
location of a suspected environmental disaster. A sample is
contacted with the array of living cells of the device (for
example, by diffusion through the matrix). The reaction of the
cells is observed indicating something about the nature of the
sample or of the living cells. As is clear to one skilled in the
art, the device of the present invention is useful for transporting
ordered cell arrays. As is clear to one skilled in the art, the
device of the present invention is also useful as an indicator or
assay device.
[0203] The method of the present invention comprises providing an
ordered array of living cells immobilized in a matrix, the matrix
configured to maintain cell viability; (b) contacting the living
cells with a stimulus; and (c) detecting the response of the living
cells to the stimulus.
[0204] To simplify detection or observation of the response, in a
preferred embodiment of the present invention, the matrix comprises
an active entity, especially an indicator, as described above. In
another preferred embodiment (alone or together with an active
entity of the matrix), the matrix is contacted with an active
entity, preferably an active entity in solution. Generally,
subsequent to contacting an active entity with the matrix, it is
necessary to wait a period of time in order to allow the active
entity to reach the proximity of the cells, for example by
diffusion.
[0205] Although there are many methods for detecting the response
of the living cells to a stimulus, the preferred method involves
the detection of light. By detection of light is meant detection of
emitted light (for example, light emitted by an indicator or light
emitted by a given cell). By detection of light is also meant
detection of light that has interacted with a given cell, the
vicinity of a given cell, or an indicator in the vicinity of a
given cell where the light is indicative of the cell response.
Clearly such detecting includes detection of fluorescence,
differential polarization and optical inspection of a cell.
[0206] A device of the present invention is a preferred device for
implementing the method of the present invention. That said, the
present invention also provides a general method for producing an
ordered array of living cells useful, for example, in implementing
the method of the present invention using a multiwell plate of the
present invention. The method of producing an ordered array of
living cells, comprises: (a) providing a multiwell plate of the
present invention, (b) placing a suspension of a plurality of
living cells in a gellable fluid in at least one well provided with
picowells; (c) causing the living cells to settle into the
picowells so as to be held in respective picowells; and (d) gelling
the gellable fluid so as to make a gel cover, trapping the living
cells between the picowells and the gel cover. In a preferred
embodiment, the picowells are made of a material comprising a gel,
preferably a hydrogel.
[0207] Generally, causing the cells to settle into the wells
includes applying a force to the cells, typical forces including
gravitation, centrifugal forces, forces resulting from the impact
of photons on the cells (e.g., laser tweezers, application of a
non-focussed laser (see, for example, P.A.L.M. Microlaser
Technologies AG, Bernried, Germany)), or forces resulting from a
pressure wave (such as produced by an ultrasonic transponder). Most
preferred is the application of centrifugal force, vide infra.
[0208] As stated above, once the cells have settled in a respective
picowell, it is preferred to gel the gellable fluid so as to form a
cover on the picowells. As a result, the cells are held snugly,
without excessive physical stress, between the inside of a
respective picowell and the surrounding gel cover. An appropriate
method of gelling a gellable fluid is dependent on the nature of
the gellable fluid and includes methods such as heating the
gellable fluid, cooling the gellable fluid, irradiating the
gellable fluid, illuminating the gellable fluid, contacting the
gellable fluid with a gelling reagent and waiting a period of time
for the gellable fluid to gel.
[0209] It is generally preferred to use a gellable fluid that forms
a hydrogel upon gelling. Exceptionally suitable gellable fluids are
fluids that comprise a material selected from the group consisting
of agars, agaroses, gelatins, low melting temperature agaroses,
alginates, room-temperature Ca.sup.2+-inducable alginates and
polysaccharides.
[0210] It is preferred that a gellable fluid that gels under
conditions that are conducive for cell survival be used for
implementing the teachings of the present invention. One preferred
gellable fluid is an alginate solution. Alginates are biologically
compatible polysaccharide proteins that are fluid at low calcium
ion concentrations (e.g., [Ca.sup.2+]<1 .mu.M) but gel upon
exposure to higher concentrations of calcium ions (e.g.,
[Ca.sup.2+]=20 mM). An exceptionally suitable alginate for
implementing the teachings of the present invention is sodium
alginate and may be purchased, for example, from Pronova
Biopolymers (Drammen, Norway) as Protanal LF120 1% in water or
Protanal LF200 1% in water.
[0211] Another preferred gellable solution is a solution of low
melting temperature agarose. Low melting temperature agaroses are
biologically compatible gels that before gelling are fluid at
temperatures that do not harm living cells (e.g., 20.degree. C.),
gel at low temperatures that do not harm living cells (e.g.,
4.degree. C.) and remain stable until well-above temperatures used
for studying living cells (40.degree. C.). An exceptionally
suitable agarose for implementing the teachings of the present
invention that may be purchased, for example, from Cambrex Bio
Science Rockland Inc. (Rockland, Me., USA) is HGS-LMP Agarose
(catalogue nr. 50221).
[0212] It is important to note that for maximal utility of a
produced device, it is desirable to ensure that substantially each
pocket holds no more than one living cell or no more than a
predetermined number of cells. This is most easily achieved by
ensuring that the picowells of the picowell array are juxtaposed
and that a given picowell is of a size to accommodate no more than
one living cell (or the predetermined number of cells). Generally
when a suspension of cells with a number of cells greater than the
number of picowells is placed in proximity of juxtaposed picowells
and the cells allowed to settle, all picowells will be filled but
there will be excess cells "stacked" on top of cells held in
picowells. A preferred method for preventing such "stacking" is
that the suspension brought in proximity of the picowells has
approximately a predetermined number of cells. It has been found
that when the number of cells in the suspension is approximately
equal to the number of picowells (or the product of the number of
picowells and the number of cells desired to be held in each
picowell), there is substantially the desired number of cells per
picowell, with only minimal stacking of cells on top of already
fully occupied picowells.
[0213] In a preferred embodiment of the method of the present
invention, the gellable fluid includes an active entity, especially
an indicator, as described above. In such a way, the active entity
becomes an integral part of the matrix of a device produced
according to the method of the present invention.
[0214] In some embodiments of the method of the present invention,
some or all of the wells of a multiwell plate are provided with
picowells. In some embodiments, all of the picowells of the
multiwell plate are substantially the same. In some embodiments,
all of the picowells in one well are substantially the same, but
are different from picowells in other wells. In some embodiments,
in a given well, there are found two different types of picowells.
By different is meant, for example, have a different size or
comprise different active ingredients.
[0215] In a specific example, a 96-well glass plate 52 of the
present invention is provided, FIG. 17. In plate 52, all wells 44
are provided with a plurality of integrally formed
hexagonally-packed knife-edged hexagonal picowells. The picowells
in each one of wells 44 of rows A, B, C and D have a diameter of 10
micron while the plurality of picowells 18 in each one of wells 44
of rows in rows E, F, G and H have a diameter of 20 micron.
[0216] Using an automatic liquid dispensing robot (Automated
Microplate Pipetting Systems--Precision.TM. XS Microplate Sample
Processor, Bio-Tek Instruments, Vinooski, Vt., USA) different
suspensions of living cells in liquid solutions of a gellable
solution, comprising a low melting temperature agarose (e.g.,
HGS-LMP Agarose of Cambrex Bio Science Rockland Inc., Rockland,
Me., USA) are added to the wells of each row. The cells suspended
in the solution dispensed in rows A, B, C and D are peripheral
lymphocytes having a diameter of about 5 to 7 microns. The cells
suspended in the solution dispensed in rows E, F, G and H are
Jurkat T cell line cells having a diameter of about 15-20. The
number of cells dispensed in each well 44 is about 95% of the
number of picowells in that well. In addition, the solutions
dispensed into a given well 44 also include active reagents:
[0217] a. in the solutions dispensed in rows A, B, E and F a first
indicator for measuring mitochondrial membrane potential is
dispensed (100 nM tetramethyl rhodamine methyl ester);
[0218] b. in the solutions dispense in rows C, D, G and H a second
indicator for measuring intracellular levels of reactive oxygen
species is dispensed (10 .mu.M dichlorodihydro fluorescein
diacetate);
[0219] Once all cell suspensions are dispensed, 96-well plate 52 is
transferred to a centrifuge provided with a cooling unit and
centrifuged so as to cause dispensed cells to settle into
picowells. After sufficient time for cell settling, centrifugation
is stopped and the cooling unit is activated so as to bring the
temperature of the gel to about 4.degree. C. and thus initiate
gelling. When sufficient time has passed for complete gelling of
the gellable fluid, the multiwell plate is used for examining
metabolism during cell death. To rows B, D, F and H a third active
agent (50 .mu.M solution of hydrogen peroxide as apoptosis inducer)
is added to each one of wells 44 of rows B, D, F and H. During the
time it takes for the third active agent to diffuse through the gel
cover, plate 52 is transferred to an observation unit configured to
detect the intensity of color developed in each picowell.
Comparison of the development of color in wells of row B (with A as
control) shows the development of mitochondrial membrane potential
as a result of apoptosis of peripheral lymphocytes. Comparison of
the development of color in wells of row C (with D as control)
shows the development of intracellular levels of reactive oxygen
species as a result of apoptosis of peripheral lymphocytes.
Comparison of the development of color in wells of row F (with E as
control) shows the development of mitochondrial membrane potential
as a result of apoptosis of Jurkat T cells. Comparison of the
development of color in wells of row H (with G as control) shows
the development of intracellular levels of reactive oxygen species
as a result of apoptosis of Jurkat T cells.
[0220] Since each of the cells is held in a respective picowell, no
cell is lost during the apoptosis process. Since the cells are held
in a substantially planar array of cells, the exact number of cells
and distribution of reactions is accurately monitored.
[0221] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination.
Although the invention has been described in conjunction with
specific embodiments thereof, it is evident that many alternatives,
modifications and variations will be apparent to those skilled in
the art. Accordingly, the present invention is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0222] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
* * * * *